Sulfation of Wnt pathway proteins

ABSTRACT

Provided is a composition comprising a peptide comprising amino acids and/or amino acid analogs comprising a continuous sequence of a sclerostin fragment comprising Tyr43 or Tyr213. Also provided is a composition comprising a peptide comprising less than about 75 amino acids and/or amino acid analogs including an amino acid or amino acid analog capable of being sulfated, where the composition is capable of inhibiting sclerostin binding to an LRP. Further provided is a composition comprising a peptide comprising less than about 75 amino acids and/or amino acid analogs including an amino acid or amino acid analog capable of being post-translationally sulfated, where the composition is capable of inhibiting binding of a protein ligand comprising a sulfation site to its binding partner. Additionally provided is a method of enhancing a Wnt signaling pathway comprising contacting an LRP5/6 receptor in the Wnt signaling pathway with either of the above-described compositions that comprise a sequence of a sclerostin fragment or is capable of inhibiting sclerostin binding to an LRP, where the tyrosine or tyrosine analog is not sulfated, in a manner sufficient to enhance the Wnt signaling pathway. Further provided is a method of treating a subject having a disease exacerbated by inhibition of a Wnt signaling pathway comprising administering either of the above-described compositions that comprise a sequence of a sclerostin fragment or is capable of inhibiting sclerostin binding to an LRP, where the tyrosine or tyrosine analog is not sulfated, to the subject in a manner sufficient to reduce the inhibition of the Wnt signaling pathway. Also, a method of inhibiting binding of a protein ligand comprising a sulfation site to its binding partner is provided. The method comprises adding the above-described composition that is capable of inhibiting binding of a protein ligand to its binding partner to the protein ligand and its binding partner in a manner sufficient to inhibit binding of the protein ligand to its binding partner.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part of prior application Ser. No.13/088,059, filed Apr. 15, 2011, which is a continuation-in-part ofapplication Ser. No. 12/802,447, filed Jun. 7, 2010.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Nov. 15, 2011, isnamed EN93CIP2.txt and is 9,424 bytes in size.

BACKGROUND OF THE INVENTION

(1) Field of the Invention

The present application generally relates to manipulation of signaltransduction proteins. More specifically, the invention is directed tosulfated Wnt pathway proteins and the manipulation of those proteins forresearch, diagnostic and therapeutic purposes

(2) Description of the Related Art

As used herein and in parent U.S. patent application Ser. Nos.13/088,059 and 12/802,447, “sulfation” or “sulfonation” is thepost-translational addition of a sulfate moiety to a protein.

Although the nature of a protein is dictated primarily by the particularamino acid sequences derived from transcription of its nucleic acidcoding sequence, there are post-transcriptional processes that may alsoaffect its properties. Some of these modifications are large scalerearrangements such as: (a) conversion of an inactive pro-enzyme into anactive form by removal of part of an amino acid sequence; (b) proteasedigestion of a composite protein into individual segments with variedfunctions as seen in some viral proteins (for instance, the polyproteinof HIV); or (c) removal of an internal amino acid sequence (an intein)by protein splicing. In addition to these cleavage processes,modification of individual amino acids can take place by enzymaticaddition of functional groups such as methyl, acetyl, phosphate,glycosyl, palmitoyl, sulfate and ubiquitin groups.

The difference in functionality caused by these modifications can induceradical differences in properties. For example, proinsulin is aninactive enzyme that is only found in its active form (insulin) afterproteolytic cleavage transforms the protein into separate peptide chainsconnected by disulfide bonds. In another instance, the addition of aubiquitin moiety does not necessarily affect its enzymatic functions butgenerates a signal for degradation of the “tagged” protein. Evenrelatively modest alterations, such as acetylation and phosphorylationof one or more amino acids in a protein, can induce remarkable changesin the properties of a protein target. The importance of both of theseprocesses in controlling levels of activities within cells by suchmodifications can be seen by the abundance of substrate specificversions of each of these family of proteins (acetylases and kinases)within a cell. Further control is exerted by the action of proteins thatreverse these changes, i.e., de-acetylases and phosphatases. Thesemodifications can result in an increase or a decrease in the activitylevel of the target protein and/or a change in its physical locale.

Although the kinase and acetylase modifications are well known areas ofresearch, the importance of sulfation is receiving increased attention.For recent reviews see Stone et al., 2009 New Biotechnology 25:299-317and Monigatti et al., 2006 Biochim Biophys Acta 1764:1904-1913.Sulfation of tyrosines is believed to take place in about 1% of thetyrosines in proteins and appears to facilitate protein-proteininteractions (Baeuerle and Huttner 1985 JBC 260:6434-6439, Kehoe andBertozzi 2000 Chem Biol 7:R57-R61). Of particular interest is theconnection between sulfation with receptors and their ligands, since theenzymes responsible for sulfation, tyrosylprotein sulfotransferase-1(TPST1) and TPST2, are localized in the Golgi apparatus. Althoughsulfation has been mostly studied in cytokine receptors and theirligands, it has been recently noted that unsulfated Wnt does notgenerate as strong a signal as sulfated Wnt, presumably due to adifferential ability of the unsulfated ligands to bind the LRP5/6receptors that are involved in the Wnt signaling system (Cha et al.,2009 Current Biol 19:1573-1580). In addition to tyrosine, evidence hasbecome available that serine and threonine are also potential sites,although at the present time it is not known if this is carried out bythe same enzymes that modify tyrosines (TPST-1 and TPST-2) or if anotherenzyme or enzymes are responsible (Medzihradszky et al., 2004 Molec CellProteomics 3:429-440). The increased binding of sulfated proteins fortheir binding partner is, at least in some cases, due to the formationof a salt bridge between the sulfate group and arginine residues on thebinding (see Woods et al., 2007, J. Proteome Res. 6:1176-1182 andreferences cited therein).

Testing for the presence of sulfation modifications in a protein can becarried out using various methods (for reviews, see Monigatti et al.2006, Stone et al. 2009, and Seibert and Sakmar 2007 Polymer90:459-477). The two most popular methods for this type of analysis isthe use of mass spectrometry (MS), or antibodies that are specific forSulfo-Tyr. With regard, to mass spectrometry, definitive answers on thepresence of sulfated tyrosines can be achieved, but due to the labilityof the bond between the sulfate group and tyrosine, specialmodifications have to be made to the standard mass spectrometryprotocols (Drake and Hortin, 2010 Int J Biochem Cell Biol 42:174-179).In a more biological approach, antibodies have been developed that candetect the presence of sulfated tyrosine residues. Antibodies have beendeveloped that can detect the presence of sulfated tyrosines regardlessof the particular peptide sequence they are embedded within (Kehoe etal., 2006 Molec Cell Proteomics 5:2350-2363; Hoffhines et al., 2006 J.Biol Chem 281:37,877-37,887). The general nature of their recognitionallows a wide variety of different proteins to be identified as long asthey contain a sulfated tyrosine. In many cases, proteins have to beisolated or separated for this type of analysis to observe individualeffects, since there is no discrimination between the different sulfatedproteins by such antibodies. For instance, the extent of sulfation canbe determined for individual isolated proteins of interest or patternsof a group of proteins can be analyzed. In an alternative approach,antibodies have been developed for specific proteins with a sulfatedtyrosine. These antibodies can detect differences between sulfated andnon-sulfated forms and can identify the presence of the sulfated proteinin a mixture of other proteins (Bundgaard et al., 2008 Methods Mol Bio446:47-66). The specificity of the epitope requires that a new antibodyhas to be developed for each particular protein of interest.

As information has accumulated concerning the amino acid sequences thatare used as substrates for sulfation, it has become clear that there isno simple consistent recognition sequence (see, e.g., Niehrs et al.,1990 JBC 265:8525-8532, Bundgaard et al., 1997 JBC 272:31,700-31,705). Acomputer program called “Sulfinator” has been created recently that iscapable of analyzing protein sequences and predicting the presence orabsence of sulfation sites (Monigatti et al. 2002 Bioinformatics18:769-770). The program achieves its highest accuracy only whenproteins are tested that are either receptors, or ligands for receptors,because these are proteins that are processed through the Golgiapparatus where the TPST-1 and TPST-2 enzymes are localized. Proteinsthat are cytosolic in nature are physiologically irrelevant since evenif they have appropriate sequences they would never come into contactwith the tyrosine sulfotransferases. The Sulfinator does not detect theextent of sulfation.

In detecting the extent of sulfation, experiments have shown that evenproteins that are substrates for sulfation do not always represent ahomogeneous population with complete sulfation. For example, gastrinpeptides, which are easily sulfated, show a mixed population of bothsulfated and unsulfated forms in roughly equal proportions (Hilsted andRehnfeld 1987 JBC 262:16,953-16,957). In another instance, there may betissue specific differentiation on the extent of tyrosine sulfation ofChromogranin A that depends upon whether it is made in parathyroid oradrenal cells (Gorr and Cohn, 1999, JBC 265:3012-3016). Differenteffects have also been observed for proteins such asgastrin/cholecystokinin peptides and their precursors where varyingdegrees of modification are seen during ontogenesis and pathogenesis ofcertain diseases (Rehfeld et al., 1989 Biochimie 70:25-31). Furthermore,in certain circumstances, such as in the expression of clonedrecombinant proteins, there may be undersulfation of proteins that wouldotherwise be completely modified (Seibert and Sakmar 2008 Biopolymers90:459-477).

Although extensive efforts have been made in searching forpharmaceutical agents that affect kinase activity, compounds that affectsulfation modifications have only recently attracted attention (see,e.g., Hemmerich et al., 2004 Drug Discovery Today 9:967-975). Thepotential utility of influencing sulfation reactions can be seen,however, by recent discoveries that CCR5, one of the receptors forrecognition of HIV, is sulfated. The importance of this modification canbe seen by results with chlorate (an inhibitor of tyrosine sulfation),where the presence of this factor decreases the affinity of gp120/CD4complexes toward the CCR5 receptor (Farzan et al., 1999 Cell96:667-676). Although there are instances where the presence of asulfation modification enhances binding, there are also numerousinstances where there is an absolute requirement for sulfation to takeplace in order for certain proteins to have biological activity (Farzanet al., 2001 J Exp Med 193:1059-1065; Costaglia et al. 2002 EMBO J21:504-513; Gao et al., 2003 JBC 278:37902-37908; Gutierrez et al., 2004JBC 279:14726-14733; Hirata et al., 2004 JBC 279:51775-51782, Fieger etal., 2005 FASEB J 19:1926-1928 and Colvin et al., 2006 Molec Cell Biol26:5838-5849).

Furthermore, in vitro studies also show the importance of sulfation withregard to binding of gp120/CD4 complexes with CCR5 peptides (Cormier etal., 2000 Proc. Nat. Acad. Sci USA 97:5762-5767). As such, it has beenrecognized that the disruption of the sulfation of CCR5 may be atreatment for HIV infection and disease processes. In another example,Liu et al. 2008 (Am J Resp Cell Molec Biol 38:738-743) hypothesized thatsulfation was a general feature of cytokine receptors and found that atleast 10 different cytokine receptors that are involved in asthma andchronic obstructive pulmonary disease (COPD) are sulfated. On thisbasis, the authors concluded that incorporation of this discovery intothe structural design of receptor antagonists might show value in thedevelopment of effective drug therapies for asthma, COPD and similarinflammatory lung diseases.

Changes in sulfation patterns have also been found for tumor derivedenzymes (Itkonen et al., 2007 FEBS Journal 275:289-301 and a dependencyon sulfation has been shown for binding of P-selectin to cancer cells(Ma and Geng 2002 J Immunol 168:1690-1696) and tumorigenesis (Feng etal., 2010 J Vir 84:3351-3361).

SUMMARY OF THE INVENTION

The present invention is based in part on the discovery that peptideshaving the amino acid sequence of small regions of sclerostin having atyrosine that can be sulfated can compete with partially sulfatedfull-length sclerostin to prevent the full-length sclerostin frominhibiting Wnt pathways.

Provided is a composition comprising a peptide comprising amino acidsand/or amino acid analogs. In these embodiments, the peptide comprises acontinuous sequence of a sclerostin fragment comprising Tyr43 or Tyr213,where the sclerostin fragment is less than about 75 amino acids.

In other embodiments, the present invention provides a compositioncomprising a peptide comprising less than about 75 amino acids and/oramino acid analogs including an amino acid or amino acid analog capableof being sulfated. In these embodiments, the composition is capable ofinhibiting sclerostin binding to an LRP.

In additional embodiments, a composition comprising a peptide comprisingless than about 75 amino acids and/or amino acid analogs including anamino acid or amino acid analog capable of being post-translationallysulfated is provided. In these embodiments, the composition is capableof inhibiting binding of a protein ligand comprising a sulfation site toits binding partner.

Additionally provided is a method of enhancing a Wnt signaling pathwaycomprising contacting an LRP5/6 receptor in the Wnt signaling pathwaywith either of the above-described compositions that comprise a sequenceof a sclerostin fragment or is capable of inhibiting sclerostin bindingto an LRP in a manner sufficient to enhance the Wnt signaling pathway.

Further provided is a method of treating a subject having a diseaseexacerbated by inhibition of a Wnt signaling pathway comprisingadministering either of the above-described compositions that comprise asequence of a sclerostin fragment or is capable of inhibiting sclerostinbinding to an LRP to the subject in a manner sufficient to reduce theinhibition of the Wnt signaling pathway.

Also, a method of inhibiting binding of a protein ligand comprising asulfation site to its binding partner is provided. The method comprisesadding the above-described composition that is capable of inhibitingbinding of a protein ligand to its binding partner to the protein ligandand its binding partner in a manner sufficient to inhibit binding of theprotein ligand to its binding partner.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the identification of sulfated tyrosines in sclerostin bymass spectrometry. The sequence at the to of FIG. 1-1 is SEQ ID NO:29;the sequence on the top of FIG. 1-2 is SEQ ID NO:8.

FIG. 2 shows results of treatment of sclerostin with TPST-1 and asubsequent comparison between treated and untreated sclerostin withregard to binding to the LRP5 receptor.

FIG. 3 shows the differential effects produced by TPST-1-treated anduntreated sclerostin with regard to Wnt-induced Alkaline Phosphataseexpression.

FIG. 4 is a comparison between epitopes defined by sulfation sites ofsclerostin and epitopes previously described in the literature. Eachsequence in FIG. 4 is SEQ ID NO:30.

FIG. 5 shows the binding of an alkaline phosphatase-LRP5 fusion proteinto sulfated sclerostin (“Normal”) vs. chlorate-treated sclerostin(“Unmodified”).

FIG. 6 shows the binding of an alkaline phosphatase-LRP5 fusion proteinto chlorate-treated sclerostin subsequently treated with PAPS only(“Unmodified”) vs. chlorate-treated sclerostin subsequently treated withTPST-1 and PAPS (“In vitro modified”).

FIG. 7 shows the binding of an alkaline phosphatase-LRP5 fusion proteinto chlorate-treated sclerostin subsequently treated with TPST-1 only(“Unmodified”) vs. chlorate-treated sclerostin subsequently treated withTPST-1 and PAPS (“In vitro modified”).

FIG. 8 shows the inhibition of Wnt activity after adding Dkk1,Dkk1+TPST-1, Dkk1+TPST-2, or Dkk1+TPST-1+TPST-2.

FIG. 9 shows (top) the amino acid sequence of sclerostin (SEQ ID NO:31),where the tyrosines (Y) that are capable of being sulfated areunderlined, and (bottom) three NMR-derived three-dimensional structuresof sclerostin (taken from Veverka et al., 2009, J. Biol. Chem.284:10890-10900).

FIG. 10 shows the inhibition of Wnt activity after adding Dkk1 preparedin insect cells that were or were not treated with chlorate.

FIG. 11 shows reversal of sclerostin-mediated Wnt inhibition bysclerostin-derived peptides having a sulfation site.

FIG. 12 shows the inhibition of sclerostin-LRP binding by two unsulfatedsclerostin-derived peptides having a sulfation site (PRN8831—having thesequence of sclerostin amino acids 198-213 where 213 is the sulfationsite, and PRN8829 [unsulfated] and PRN8830 [sulfated], each having thesequence of sclerostin amino acids 37-52 where 43 is the sulfationsite), including treatments where PRN8831 is sulfated with treatment bythe sulfating enzymes TPST1 and/or TPST2.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the singular forms “a”, “an” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise. Additionally, the use of “or” is intended to include“and/or”, unless the context clearly indicates otherwise.

The present invention is based in part on the discovery that several Wntpathway proteins, including sclerostin, Disheveled1 (Dvl1), Dickkopf1(Dkk1), Kremen1 (Kr1), Frizzled6 (Fz6) and LRP5. Previously, only Wnt11,Wnt5a, Wnt3a and sFRP-1 were identified as Wnt pathway proteins that aresulfated (Cha et al., 2009, Curr. Biol. 19:1573-1580; Zhong et al.,2007, J. Biol. Chem. 282:20523-20533).

An additional discovery disclosed herein is that peptides having theamino acid sequence of small regions of sclerostin having a tyrosinethat can be sulfated inhibit binding of sclerostin to LRP and competewith partially sulfated full-length sclerostin to prevent thefull-length sclerostin from inhibiting Wnt pathways. See, e.g., Example12.

As further elaborated below, the discoveries disclosed herein enablesthe use of various therapeutic, diagnostic and research methods andcompositions.

Sclerostin, a ligand of various LRP receptors, can be sulfated in atleast two different sites, Tyr₄₃ and Tyr₂₁₃ (using the UniProtKBAccession No. Q9BQB4 of unprocessed sclerostin as reference points). Asshown in the Examples below, ex vivo sulfation treatment of apreparation of recombinant sclerostin results in an increase in theaffinity of the sclerostin to the LRP5/6 receptor, as well as anincrease in its ability to suppress Wnt induced expression of alkalinephosphatase.

Various means may be employed to determine the presence of sulfationmodifications in proteins of interest. As described in Example 2 below,mass spectrometry (MS) analysis was carried out using sclerostin thatwas expressed in mammalian cells that are capable of carrying outpost-synthetic modifications such as sulfation. It should be noted thatthe standard conditions that are usually employed in MS studies leads toa rapid loss of sulfate groups. As such, when detection of sulfatedtargets is desired, avoidance of acidic conditions and lower energyinputs are required in order to increase the sensitivity of detection ofsulfated tyrosines in specimens of interest (Drake and Hortin, 2010).This type of analysis may lead to the identification of the presence ofsulfated tyrosines and, in many cases, the exact position of thesulfated amino acid. A caveat to be considered is that the simultaneouspresence of both sulfated and unsulfated tyrosines for a given fragmentdoes not give any estimate for their relative proportions prior toanalysis since the process is still liable to losses of sulfatemoieties, thereby generating some unsulfated tyrosines de novo.Distinguishing between pre-existing and converted unsulfated tyrosinesis problematic and as such, MS serves best as a qualitative tool forwhether sulfation occurs at all at a given site.

Prior to carrying out the MS analysis, some of the sclerostin was usedin a reaction with TPST-1 (Example 1) such that if any tyrosinemodification sites were present in the sample they could be convertedinto the sulfated from. As described in the MS analysis provided inExample 2, the presence of sulfation modifications was found in both thetreated and untreated samples of sclerostin, indicating that therecombinant sclerostin being tested had undergone sulfationmodifications prior to secretion from the cells used for recombinantexpression. As noted above, however, the MS analysis can determine thepresence of sulfation modifications, but is unable to provideinformation on whether there is complete or partial modification on thesulfation sites. A protein having the appropriate sulfation sequence maybe a candidate for post-synthetic modification as it passes through theGolgi apparatus prior to secretion outside of a cell, but recombinantexpression systems are essentially abnormal states and there may beundersulfation of sites that normally would be fully converted. Inaddition, there may be differences in recognition and/or efficiency whenmammalian proteins are expressed in non-mammalian systems such as insectcells.

As such, treated and untreated sclerostin was used in biological assaysto investigate whether there were any differences in the properties ofsclerostin after an in vitro treatment. As described in Example 3 and asshown in FIGS. 2 and 3, the differences seen with the in vitro treatmentare consistent with a conclusion that some tyrosines in the sulfationsites of the recombinant sclerostin were not sulfated prior to secretionfrom cells, thereby making them available for in vitro sulfation. Thefully sulfated sclerostins displayed an increased affinity for theirbinding partners, i.e., the original sample contains partially sulfatedsclerostin and the treated sample has an increased level of sulfation.This effect could likely be seen more dramatically if conditions wereused such that a comparison was made with starting material that wascompletely or mostly lacking in sulfation modifications prior to an invitro reaction. Ways that this could be accomplished are the use ofyeast or bacterial expression systems, treatment of recombinantexpression cells with chlorate prior to harvesting the protein, orexpression in cells that have been mutated to eliminate TPST activity,such as those described by Westmuckett et al., 2008, Gen. Compar.Endocrine 156:145-153. With regard to the chlorate treatment, it hasbeen previously shown that such treatment can strongly reduce the degreeof sulfation in cells (Baeuerle and Huttner 186 BBRC 141:870-877; Hortinet al., 1988 BBRC 150:342-348; Mintz et al., 1994 J Biol Chem269:4845-4852) and a bacterial or yeast host would lack any sulfationsince they intrinsically lack the sulfotransferases responsible fortyrosine sulfation (Kehoe and Bertozii 2007 Chemistry & Biology7:R57-R61). In addition to chlorate, sulfotransferase activity can beinhibited by sphingosine (Kasinathan et al., 1993, Biochemistry32:1194-1198), sulfate analogs that inhibit ATP-sulfurylase, or selenite(Stone et al., 2009, New Biotechnol. 25:299-317). Conversely, ifdesired, sulfotransferases can be enhanced by sofalcone (Kasinathan etal., 1994, Gen. Pharmacol. 25:1017-1020).

A further method of investigation is the use of a software programcalled Sulfinator that can predict the presence of a sulfation site with98% accuracy from the amino acid sequence alone (Stone et al., 2009 NewBiotechnology 25:299-317). When the sequences from sclerostin wereanalyzed with this program, it successfully identified the aminoterminal modification of sclerostin at Tyr₄₃ detected by MS analysis butmissed the carboxy terminal modification at Tyr₂₁₃. This failure inSulfinator predictability is likely due to the tyrosine in sclerostinthat is modified at the carboxy end of sclerostin being the terminalamino acid itself; since the Sulfinator program uses the neighboringsequences surrounding a tyrosine for evaluating its likelihood of beingsulfated, and by definition, a terminal tyrosine of sclerostin wouldintrinsically lack sequences on one side. It should be pointed out thatalthough the presence of a site predicted to be a sulfation site islikely dependable, there are incidents where sulfation modificationswere unrecognized by the Sulfinator program but later identified inphysical studies (Onnerfjord et al., 2004 JBC 279:26-33, Hoffhines etal., 2006 JBC 281:37877-37887). Nonetheless, the recognition of theTyr₄₃ modification by the Sulfinator program is an independentconfirmation of the sulfation of this particular amino acid insclerostin.

It should be understood that although sulfated tyrosines have beenobserved in many secreted ligands and their receptors, their presence isnot necessarily required and it is inappropriate to make any predictionsabout their presence in the absence of any investigational analysis. Asnoted above, it has been estimated that ˜1% of the tyrosines in cellularproteins are modified tyrosines (Huttner 1984 Meth Enzymol 107:200-223)which in turn has the implication that ˜99% of them would not have thismodification.

As shown in Example 9, the use of the Sulfinator program resulted inpositive predictions of a sulfonation site in Dishevelled (Dvl)1 but notin Dvl2. However, since the Dishevelled protein is an intracellularprotein, it may not come into contact with the Golgi apparatus and thein vivo significance of the site in Dvl 1 is not readily apparent.However, it does show that not every protein that is tested with theSulfinator program automatically comes up with a positive result.

The same phenomenon is seen with regard to the Dkk family. Although all4 members were tested, the presence of a sulfation site was predicted toonly be present in human and mouse Dkk1 while neither human nor mouseDkk2, Dkk3 or Dkk4 were predicted to have sulfation sites. The presenceof such a site in Dkk1 is of interest in a number of different regards.For instance, in many cases the properties of Dkk1 seems to be theopposite of Dkk2 and Dkk4, where intact Dkk1 is regarded as a strongrepressor of Wnt induced stimulation whereas under some circumstancesDkk2 and Dkk4 can enhance Wnt induced activity. It is possible thatdifferential sulfation may be a factor in this separation of properties.Secondly, as described with regard to the sulfation site in sclerostin,the discovery of the presence of a sulfation moiety in Dkk1 implies thata virtual screening program that employs the structure of Dkk1 may bemade more accurate by including the influence of the sulfationmodification when predicting binding affinities of small molecules toDkk1. Lastly, the presence of the sulfation modification endows Dkk1with a previously unknown epitope that may be used in conjunction withan antibody that binds to the sulfation site.

A number of different members of the Frizzled family were also testedwith the Sulfinator program including Fz1, Fz2, Fz3, Fz4, Fz5, Fz6, Fz7mFz8, Fz9 and Fz10 proteins. The results of these tests were a lack ofidentification of a predicted sulfation site in the majority of theseFrizzled proteins even though they are large transmembrane proteins withnumerous tyrosines present. In the few instances where potentialsulfation sites were identified, they were only on the intracellularportion and not involved in extracellular protein/protein interactions.

Also tested were LRP5, LRP6 and LRP4. When human and mouse sequenceswere examined, Sulfinator identified a potential sulfonation site on theextracellular portion of only LRP5. For LRP 4, no sites were predictedto be present and both LRP5 and mouse LRP6 showed a potentialsulfonation site being present in the intracellular portion of thesereceptor proteins. Although this location precludes interaction withextracellular ligands, once a ligand has been bound to a receptor, thereare one or more protein/protein interactions within the cell that ispart of the signal generation process and these events may potentiallybe influenced by the presence of a sulfation modification. Theextracellular portion of LRP5 that has been identified as a sulfationsite (SEQ ID NO:26) is in the second YWTD domain, a portion of the LRP5receptor that may be involved in binding Wnt, Dkk and sclerostinligands. The influence of a sulfation modification is of value incarrying out virtual screening processes for the interaction of thesecond YWTD domain (SEQ ID NO:26) with small molecules that affectprotein/protein interactions at this site. It also has value inidentifying molecules that may discriminate between the two closelyrelated LRP5 and LRP6 receptors.

The discovery that Wnt pathway proteins, i.e., proteins that participatein the Wnt signaling system, have sulfated amino acids offers uniquemethods of analysis as well as therapeutic means. As described invarious patent applications (e.g., U.S. patent publications2005/0196349; 2006/0030523; 2008/0119402, hereby incorporated byreference), compounds that block the interaction between LRP5/6receptors and the soluble ligands Dkk and sclerostin can offer a varietyof useful therapeutic means. Since it has now been discovered that theamino acid sequence of several Wnt pathway proteins can also comprise apost-synthetic sulfation modification, compounds that have beenpreviously tested for effects on sclerostin and Dkk with regard to Wntsignaling may be retested using separate reaction mixtures or bindingassays where either the modified or unmodified versions of theseproteins are tested separately. As has been pointed out earlier, someproteins exist as a mixture of sulfated and unsulfated forms andprevious experiments may have been based upon such a mixture, withoutrecognition that the net effects might be a composite of the individualeffects on modified and unmodified Wnt pathway proteins. Control overthe particular form (sulfated or unsulfated) of the proteins now allowsinvestigation into whether compounds are more or less effective withregard to using sulfated or unsulfated versions of the proteins inassays. The lack of recognition of the potential presence of a mixtureof different forms also allows for the possibility that some effectivecompounds may have been missed due to the use of protein preparationsthat had a preponderance of one form over another.

Furthermore, the presence of a site that is involved in protein-proteininteractions is in itself a potential therapeutic target. Thus, a seriesof compounds can be surveyed to see if they specifically interrupt invitro or in vivo sulfate modification of the tyrosines or othersulfation sites in the proteins. Such pharmaceutical agents would havethe potential for modifying the level of activity induced by the proteinby controlling the degree of sulfation and thereby their affinity inprotein-protein interactions. Pharmaceutical reagents that may be usedto disrupt sulfation processes can include but not be limited to smallmolecules, peptides, organic molecules, cyclic molecules, heterocyclicmolecules, lipids, charged lipids, glycolipids, polar lipids, non-polarlipids and aptamers. The compounds may be ones that have been designedto bind to the surface of the protein through a virtual screeningprocess as described for sclerostin in US Patent Publication2005/0196349. In this process, a revised virtual structure of theprotein may be devised to takes into consideration the presence of thesulfation of amino acids. Contrariwise, compounds may be testedindependently from virtual screening and tested strictly on a randombasis or they may be selected to have a physical resemblance tocompounds that result from virtual screening processes. Such a processcan also include the use of mutational substitutions at the modificationsites (see, for instance, U.S. Patent Publication 2005/0196349). Thus, aseries of (selected or random) compounds may be assayed for an abilityto eliminate or reduce sulfation of the protein, by any means that havepreviously been described for analysis and/or detection of sulfation ofproteins. As a control, one or more proteins that also have sulfationsites may be included to insure that the blockage of sulfation isspecific for the target of interest. Any means that have been describedin the past for detection of the presence of sulfated tyrosines may beused in this aspect of the present invention, thus for example, thesemeans may be as complex as carrying out MS analysis to simpler methodssuch as incorporation of ³⁵S PAPS by TPST, immunoassays that useantibodies that recognize proteins with sulfated tyrosines irrespectiveof their context (Kehoe et al., 2006 and Hoffhiner et al., 2006, J Biol.Chem. 281:37877-87), or antibodies that are specific for the sulfated orunsulfated forms of the protein (as discussed in more detail below). Ifdesired, truncated versions of the protein that comprise the target areaof interest may also be used as substrates in assays as long as theirbiological structures/functions are similar to the parent protein. Inaddition, peptides that may represent the sulfation site of the proteinmay also be used.

Investigations into compounds that might interrupt sulfation of proteinshas been previously described by Hortin et al., 1988 BBRC 150:342-348where compounds were found that were non-specific in that they inhibitedsulfation of proteins, oligosaccharides and proteoglycans (although withvarying efficiencies). A similar study has been done more recently byKehoe et al., 2002 (Bioorg Med Chem Letters 12:129-132) where twocompounds were identified that inhibited sulfation by TPST-2. Similar tothe results published earlier by Hortin et al., further testing showedthat the inhibitors affected other sulfotransferases as well. Even ifthese inhibitors only affected the TPST reaction itself, however, thisapproach would indiscriminately block sulfation of a wide variety ofdifferent protein targets and thereby lead to potentially toxic effects.It should be noted that knockout mice lacking either TPST-1 (Ouyang etal., 2002 JBC 277:23,731-23,787) or TPST-2 (Borghei et al., JBC281:9423-9431) activity are essentially viable but exhibit a variety ofpleiotropic defects. Partial overlap in the functionality of the twoTPST enzymes can be seen by experiments with a double knockout that ismissing both TPST-1 and TPST-2 where most progeny died soon after birthand any survivors failed to thrive (Westmuckett et al., 2008). Thesedouble knockouts exemplify a situation that may be more akin to thepresence of a general TPST inhibitor. In addition, as noted previously,there are many proteins involved in protein-protein interactions wheresulfation is obligatory for biological activity and some are involved ininflammatory responses that require sulfation for functionality; assuch, it may be that the double knockouts are phenotypically silentexcept under certain conditions where such responses would be induced orrequired. Targeting the modification of a particular sulfation target asdescribed in the present invention is likely to be superior to effortsto block sulfation in general since it is likely to have more specificeffects than a general blockage that may produce deleterious as well asbeneficial effects.

As indicated above, prokaryotic expression systems lack the ability topost-translationally modify proteins expressed therein. As such, a Wntpathway protein having a sulfation site, when recombinantly expressed inprokaryotes such as bacteria (e.g., E. coli) are not sulfated orglycosylated, even though such proteins are sulfated and glycosylatedwhen expressed naturally or in eukaryotic expression systems. Thus, if aWnt pathway protein having a sulfation site is expressed in aprokaryotic system to achieve the high yields that can be obtained fromsuch expression systems, the protein will not be sulfated orglycosylated. Such a protein can then be sulfated, e.g., by using TPST,to obtain in high yield a Wnt pathway protein that is sulfated but notglycosylated. The TPST treatment can be achieved in vitro or in a cellexpressing a TPST, either as a native enzyme or produced recombinantly.

Thus, provided herein is a composition comprising an isolated andpurified Wnt pathway protein, where the protein is sulfated but notglycosylated. In various embodiments, the protein is sulfated on atyrosine. The Wnt pathway protein in these embodiments can be from anysource, for example an insect (e.g., a Drosophila), an amphibian (e.g.,a Xenopus), or a mammal (e.g., a rodent or human).

In some embodiments, the Wnt pathway protein is sulfated at a nativesulfation site. As used herein, a “native sulfation site” is an aminoacid sequence of a protein that would ordinarily be sulfated whenexpressed in a cell where the protein would normally be found. Here, theWnt pathway protein can be any protein that has a native sulfation site,e.g., a Wnt, for example Wnt5A, Wnt11, Wnt3a or another Wnt having anative sulfation site. The protein can also be, e.g., a sclerostin, aDvl1, a Dkk1, a Kr1, a Fz6, an LRP5, or any other Wnt pathway proteinnow known or later discovered to comprise a native sulfation site. Wherethe protein is a sclerostin, in various embodiments the protein issulfated on the tyrosine equivalent to the Tyr₄₃ or Tyr₂₁₃ of humansclerostin (i.e., the corresponding position when the sclerostin is nothuman sclerostin). Where the protein is a Dvl1, the protein in variousembodiments is sulfated on the tyrosine equivalent to the Tyr₈ of humanDvl1. Where the protein is Dkk1, in various embodiments the protein issulfated on the tyrosine equivalent to the Tyr₈₃ of human Dkk1. Wherethe protein is a Kr1, in various embodiments the protein is sulfated onthe tyrosine equivalent to the Tyr₁₇₅ or the Tyr₁₇₈ of human Kr1. Wherethe protein is Fz6, in various embodiments, the protein is sulfated onthe tyrosine equivalent to the Tyr₅₈₀ of human Fz6. Where the protein isLRP5, in various embodiments the protein is sulfated on the tyrosineequivalent to the Tyr₃₈₀ or the Tyr₁₅₈₃ of human LRP5.

In other embodiments, the protein is sulfated at a sulfation site thatis not native to the protein. Such a sulfation site can be added to thenative protein by any means, including by recombinant DNA methods, or bychemical methods. In various embodiments, the sulfation site comprises atyrosine that is not native to the protein. In other embodiments, thetyrosine is native to the protein but surrounding amino acids aremodified to engineer a sulfation site that is recognized by asulfotransferase, e.g., a TPST enzyme. The protein of these embodimentscan be any protein in the Wnt pathway, including a Wnt, a Dvl2, a Dvl3,a Dkk2, a DKK3, a DKK4, a Kr2, a Fz1, a Fz2, a Fz3, a Fz4, a Fz5, a Fz7,a Fz8, a Fz9, a Fz10, an LRP4, an LRP6, or any other Wnt pathwayprotein, including proteins that have a native sulfation site andproteins that do not have a native sulfation site, including GSK-3β,APC, β-Catenin, Axin, TCF, LEF, or any other Wnt pathway protein nowknown or later discovered.

It is expected that a TPST enzyme from any species would cause sulfationof a sulfation site on a protein from any species.

With the discovery disclosed herein that many Wnt pathway proteinscomprise a sulfation site, and that in natural eukaryotic systems only aproportion of Wnt pathway proteins that comprise a sulfation site areactually sulfated, it becomes clear that a preparation where all the Wntpathway proteins are either sulfated or not sulfated is desirable. Thus,where a Wnt pathway protein is prepared in a eukaryotic system, it isnow clear that glycosylated protein that is a mixture of sulfated andunsulfated forms is obtained if measures are not taken to obtain onlysulfated protein (for example by treating the protein with TPST) orunsulfated protein (for example by preparing the protein in cellsexposed to chlorate).

Thus, provided is a preparation of a Wnt pathway protein comprising atleast one sulfation site and at least one glycosylation site, where allof the Wnt pathway protein in the preparation is glycosylated but notsulfated. In some embodiments, the Wnt pathway protein does not comprisea native sulfation site, but the sulfation site is engineered into theprotein, as described above. In other embodiments, the Wnt pathwayprotein has a native sulfation site. The preparation can be achieved byany means known in the art, for example by translating the protein in aeukaryotic cell treated with a compound that inhibits sulfation. See,e.g., Stone et al., 2009, New Biotechnol. 25:299-317. Hortin et al.,1988 BBRC 150:342-348, and Kehoe et al., 2002, Bioorg Med Chem Letters12:129-132. In some embodiments, the compound that inhibits sulfation ischlorate. The Wnt pathway protein in these embodiments can be from anysource, for example an insect (e.g., a Drosophila), an amphibian (e.g.,a Xenopus), or a mammal (e.g., a rodent or human). The Wnt pathwayprotein in these embodiments can be any such protein that has a nativesulfation site, e.g., a Wnt, for example Wnt5A, Wnt11, Wnt3a or anotherWnt having a native sulfation site. The protein can also be, e.g., asclerostin, a Dvl1, a Dkk1, a Kr1, a Fz6, an LRP5, or any other Wntpathway protein now known or later discovered that comprises a nativesulfation site.

As indicated above, a Wnt pathway protein can be engineered to comprisea sulfation site that is not present in the native Wnt pathway protein.Such a protein would be expected to increase binding to its nativebinding partner if the protein is engineered such that the nonnativesulfation site mimics a sulfation site present in analogs of the Wntpathway protein. For example, as shown in Example 9, human Dkk1 has asulfation site comprising a tyrosine at position 83, but human Dkk2 doesnot comprise a sulfation site. The human Dkk2 can be engineered to havea sulfation site by modifying the region in that protein thatcorresponds to the region around position 83 of human Dkk1 to have thesame amino acid sequence as the human Dkk1 protein (i.e., DNYQPYPCAEDE(SEQ ID NO:1)). Such a modified Dkk2 would be sulfated like Dkk1 andwould be likely to have increased binding to LRP5/6 and increased Wntinhibitory activity similar to Dkk1 (see Example 10). Similarly, asulfation site can be engineered into a region of a Wnt pathway proteinfrom one species by modifying a region in that protein that correspondsto a region of the homolog from another species that has a sulfationsite. For example, as shown in Example 9, mouse LRP6 has a sulfationsite comprising a tyrosine at position 1562, but the human LRP6 does nothave a sulfation site. The human LRP6 can be engineered to modify theregion around the human LRP6 corresponding to the region of the mouseLRP6 around position 1562. Such a sulfated human LRP6 would be expectedto behave similar to the sulfated mouse LRP6.

Thus, the present invention provides a modified Wnt pathway proteincomprising a sulfation site that is not present in the native Wntpathway protein. The Wnt pathway protein in these embodiments can befrom any source, for example an insect (e.g., a Drosophila), anamphibian (e.g., a Xenopus), or a mammal (e.g., a rodent or human). Insome embodiments, the Wnt pathway protein further comprises a nativesulfation site. In other embodiments, the Wnt pathway protein does notfurther comprise a native sulfation site. Nonlimiting examples ofproteins that can be utilized in these embodiments is a Wnt, is asclerostin, a Dvl1, a Dkk1, a Kr1, a Fz6, an LRP5, a Dvl2, a Dvl3, aDkk2, a DKK3, a DKK4, a Kr2, a Fz1, a Fz2, a Fz3, a Fz4, a Fz5, a Fz7, aFz8, a Fz9, a Fz10, an LRP4, an LRP6, or any other Wnt pathway proteinnow known or later discovered. The protein of these embodiments can beprepared by recombinant DNA methods or by chemical methods.

Similar to the above embodiments, the present invention also provides amodified Wnt pathway protein lacking a sulfation site that is present inthe native Wnt pathway protein. Such a protein is useful where reducedbinding of the Wnt pathway protein is desired. These proteins can beprepared by any of a number of strategies, e.g., by engineering theprotein to eliminate the tyrosine that is sulfated in the nativeprotein, or by engineering the sulfation site to be the same as ahomologous protein from a different species, or from the same family(for example by engineering the region around position 83 in human Dkk1to have the same sequence as the analogous region of human Dkk2). Thesemodified proteins can be prepared by recombinant methods or by chemicalmethods. Nonlimiting examples of proteins that can be utilized in theseembodiments is a Wnt, is a sclerostin, a Dvl1, a Dkk1, a Kr1, a Fz6, anLRP5, a Dvl2, a Dvl3, a Dkk2, a DKK3, a DKK4, a Kr2, a Fz1, a Fz2, aFz3, a Fz4, a Fz5, a Fz7, a Fz8, a Fz9, a Fz10, an LRP4, an LRP6, or anyother sulfation site-containing Wnt pathway protein now known or laterdiscovered. The protein of these embodiments can be prepared byrecombinant DNA methods or by chemical methods.

Nucleic acids comprising a nucleotide sequence encoding these modifiedWnt pathway proteins is also provided, as are vectors (e.g., bacterial,yeast, mammalian, viral, expression, shuttle, plasmid, etc.) comprisingthe nucleic acid. Where the vector is an expression vector, the vectorcan further comprise control elements such that the modified protein isexpressed constitutively or under the control of an inducible promoter.Prokaryotic and eukaryotic cells comprising these vectors are alsoenvisioned. These cells and vectors can be administered or implantedinto a mammal, e.g., a rodent or a human, e.g., for therapeutic purposes

The identification of peptide sequences comprising a modified tyrosinealso allows the use and design of artificial peptides that contain thesemodifications. Presumably these should have higher binding affinitiesthan their unmodified counterparts. In this regard, it is noted thatpeptides comprising a sulfated tyrosine can mimic the binding of thesulfated whole protein from which it was derived. See, e.g., Farzan etal., 2001, J. Exp. Med. 193:1059-1065. Although the sulfated peptidewould be the basis for the design, it is understood that the actualcomponents can be artificial equivalents of these peptides. Examples ofcompounds made with such components can comprise but not be limited tothe peptide mimetics described in pending U.S. Patent Publication2008/0119402, as well the substitution of dextro isomers instead of thenormal levo forms and peptidomimetics such as those described in Hammondet al., 2006 Chem & Biol 13:1247-1251. Other examples of analogs thatmay find use with the present invention are “unnatural amino acids”where in it is understood that in the context of the present invention“unnatural amino acids” refers to amino acids that are not geneticallyencoded, i.e., they are not represented by a nucleotide codon. Thiswould include the dextro isomers discussed above as well as other aminoacids such as Aib (amino-isobutyric acid), bAib (3-aminoisobutyricacid), Nva (norvaline), β-Ala, Aad (2-amino-adipic acid), bAad(3-aminoadipic acid), Abu (2-aminobutyric acid), Gaba (γ-aminobutyricacid), Acp (6-aminocaproic acid), Dbu (2,4-diaminobutyric acid), TMSA(trimethylsilyl-Ala), alle (allo-Isoleucine), Nle (Norleucine),tert.Leu, Cit (Citrulline), Orn, Dpm (2,2′-diaminopimelic acid), Dpr(2,3-diaminopropionic acid), α- or β-Nal, Cha (cyclohexyl-Ala),hydroxy-proline, Sar (Sarcosine) etc., cyclic amino acid units andN^(α)-alkylated amino acid units, e.g. MeGly (N^(α)-Methyl-glycine),EtGly (N^(α)-ethylglycine) and EtAsn (N″-ethyl-asparagine). Accordingly,synthetic peptides can be made that include one or more of theseunnatural amino acids. See, e.g., Dong, U.S. Patent Publication2007/0299009 and Stevenson, 2009, Curr. Pharm. Biotechnol. 10:122-137for examples of useful peptides comprising amino acid analogs.

Thus, further provided herein is a composition comprising a peptide lessthan 75 amino acids or amino acid analogs. In these embodiments, thepeptide consists of a fragment of a Wnt pathway protein, where thefragment is sulfated. The peptide can be, e.g., at least 5 amino acidsor amino acid analogs long, at least 10 amino acids or amino acidanalogs long, at least 20 amino acids or amino acid analogs long, atleast 30 amino acids or amino acid analogs long, at least 40 amino acidsor amino acid analogs long, at least 50 amino acids or amino acidanalogs long, at least 60 amino acids or amino acid analogs long, or atleast 70 amino acids or amino acid analogs long. In some embodiments,the peptide is sulfated at a sulfation site native to the Wnt pathwayprotein. In other embodiments, the peptide is sulfated on an amino acidor amino acid analog that is not subjected to sulfation in the nativeprotein. The peptides can be prepared by chemical methods. See, e.g.,Seibert and Sakmar 2007, Peptide Science 90:459-477. The Wnt pathwayprotein from which the peptide is derived in these embodiments can befrom any source, for example an insect (e.g., a Drosophila), anamphibian (e.g., a Xenopus), or a mammal (e.g., a rodent or human).

The peptide of these embodiments can be sulfated by any method now knownor later discovered, for example by chemical peptide synthesis methods,or using a sulfotransferase, e.g., a TPST1 or TPST2.

The peptide can be derived from any Wnt pathway protein, for example aprotein having a native sulfation site or a protein that is engineeredto have such a site. Nonlimiting examples of proteins from which thepeptide can be derived are a sclerostin, a Dvl1, a Dkk1, a Kr1, a Fz6,an LRP5, a Wnt, a Dvl2, a Dvl3, a Dkk2, a DKK3, a DKK4, a Kr2, a Fz1, aFz2, a Fz3, a Fz4, a Fz5, a Fz7, a Fz8, a Fz9, a Fz10, an LRP4, an LRP6or any other Wnt pathway protein now known or later discovered. Activepeptides of Wnt pathway proteins are known in the art. See, e.g.,Gregory et al., 2005, J. Biol. Chem. 280:2309-2323. Where the protein isa sclerostin, in various embodiments the peptide is sulfated on thetyrosine equivalent to the Tyr₈₃ of human sclerostin. Where the proteinis a Dvl1, the peptide in various embodiments is sulfated on thetyrosine equivalent to the Tyr₈ of human Dvl1. Where the protein isDkk1, in various embodiments the peptide is sulfated on the tyrosineequivalent to the Tyr₈₃ of human Dkk1. Where the protein is a Kr1, invarious embodiments the peptide is sulfated on the tyrosine equivalentto the Tyr₁₇₅ or the Tyr₁₇₈ of human Kr1. Where the protein is Fz6, invarious embodiments, the peptide is sulfated on the tyrosine equivalentto the Tyr₅₈₀ of human Fz6. Where the protein is LRP5, in variousembodiments the peptide is sulfated on the tyrosine equivalent to theTyr₃₈₀ or the Tyr₁₅₈₃ of human LRP5.

These embodiments also encompass analogs of the above peptides having asequence that is altered from the native Wnt pathway protein, e.g.,having one or several amino acids different from the native protein,where the altered amino acids do not affect the activity of the peptide.Identification of such analogs for any peptide derived from any Wntpathway protein is routine in the art, for example by makingconservative amino acid substitutions, particularly in regions of theprotein that are not involved in interactions with an LRP, or bysubstituting amino acids or groups of amino acids of a sclerostinsequence from one species with analogous regions from another sclerostinsequence.

Thus, the present invention provides a composition comprising a peptidecomprising amino acids and/or amino acid analogs, where the peptidecomprises a continuous sequence of a sclerostin fragment comprisingTyr43 or Tyr213, wherein the sclerostin fragment is less than about 75amino acids. In various embodiments, the sclerostin fragment is lessthan about 50 amino acids, less than about 30 amino acids, less thanabout 20 amino acids, less than about 15 amino acids, less than about 10amino acids, less than about 8 amino acids, less than about 6 aminoacids, or less than about 4 amino acids. In some of these embodiments,the peptide comprises a tyrosine or tyrosine analog analogous to Tyr43of sclerostin; in other embodiments, the peptide comprises a tyrosine ortyrosine analog analogous to Tyr213 of sclerostin.

Nonlimiting examples of amino acid analogs are discussed above andinclude for example peptide mimetics described in pending U.S. PatentPublication 2008/0119402, as well the substitution of dextro isomersinstead of the normal levo forms and peptidomimetics such as thosedescribed in Hammond et al., 2006 Chem & Biol 13:1247-1251; unnaturalamino acids such as dextro isomers as well as other amino acids such asAib (amino-isobutyric acid), bAib (3-aminoisobutyric acid), Nva(norvaline), β-Ala, Aad (2-amino-adipic acid), bAad (3-aminoadipicacid), Abu (2-aminobutyric acid), Gaba (γ-aminobutyric acid), Acp(6-aminocaproic acid), Dbu (2,4-diaminobutyric acid), TMSA(trimethylsilyl-Ala), alle (allo-Isoleucine), Nle (Norleucine),tert.Leu, Cit (Citrulline), Orn, Dpm (2,2′-diaminopimelic acid), Dpr(2,3-diaminopropionic acid), α- or β-Nal, Cha (cyclohexyl-Ala),hydroxy-proline, Sar (Sarcosine) etc., cyclic amino acid units andN^(α)-alkylated amino acid units, e.g. MeGly (N^(α)-Methyl-glycine),EtGly (N^(α)-ethylglycine) and EtAsn (N^(α)-ethyl-asparagine).

In some embodiments, the composition comprises more than one (e.g. 2, 3,4, 5, 10, or more) peptide bound together, either covalently ornon-covalently, for any purpose. The peptides in these complexes can allbe the same, can be different, or a mixture of the same and differentpeptides. Additionally or alternatively, the composition can compriseother compounds, either separate from the peptides or covalently ornon-covalently bound to the peptides, also for any purpose. Nonlimitingexamples of such compounds include detectable moieties such asradioactive, electron dense, fluorescent, phosphorescent,chemiluminescent, chromogenic, chelating, magnetic, energy transfer orintercalating compounds, or nucleic acids, nucleic acid analogs,proteins, peptides, antibodies, antibody fragments, carbohydrates,polysaccharides, oligosaccharides, lipids, nucleotides, nucleotideanalogs, haptens, or organic compounds, e.g. less than 2000 daltons,1000 daltons, 500 daltons or 250 daltons.

The sclerostin from which these peptides are derived can be anyeukaryotic sclerostin now known or later discovered, includinginvertebrate or vertebrate sclerostin, e.g., a mammalian sclerostin suchas a human sclerostin.

The peptides of these or any of the other embodiments disclosed hereincan be prepared by any method known in the art, for example byrecombinant DNA methods or by chemical synthesis methods.

In some of these compositions, the peptide is sulfated at the amino acidor amino acid analog corresponding to Tyr43 or Tyr213 of sclerostin. Thepeptide of these embodiments can be sulfated by any method now known orlater discovered, for example by chemical peptide synthesis methods, orusing a sulfotransferase, e.g., a TPST1 or TPST2.

In additional embodiments of these compositions, the peptide is notsulfated at the amino acid or amino acid analog corresponding to Tyr43or Tyr213 of sclerostin.

Preferably, the sulfated or unsulfated peptide is capable of reducingsclerostin inhibition of a Wnt pathway, for example as described inExample 12 below.

Also provided is a composition comprising a peptide comprising less thanabout 75 amino acids and/or amino acid analogs where the peptideincludes an amino acid or amino acid analog capable of being sulfated.In these embodiments, the composition is capable of inhibitingsclerostin binding to an LRP.

In some of these embodiments, the peptide comprises a sequence at leastabout 50% homologous to a continuous sclerostin fragment comprisingTyr43 or Tyr213, where the peptide comprises a tyrosine or tyrosineanalog at the position analogous to the Tyr43 or Tyr213 of humansclerostin.

The skilled artisan could easily identify a multitude of the peptides ofthese embodiments that are not completely homologous with a sclerostin,for example by making conservative amino acid substitutions in awild-type sclerostin sequence, or by substituting amino acids or groupsof amino acids of a sclerostin sequence from one species with analogousregions from another sclerostin sequence.

In various embodiments, the peptide has at least about 60%, 70%, 80%,85%, 90%, 95%, or 98% homology to the sclerostin fragment. In otherembodiments, the sclerostin fragment is less than about 50, 30, 20, 15,10, 8, 6 or 4 amino acids.

As in the peptides in the compositions described above, the tyrosine ortyrosine analog can be sulfated or unsulfated. In preferred embodiments,the composition is capable of reducing sclerostin inhibition of a Wntpathway.

The discovery that small peptide fragments of sclerostin that comprise atyrosine sulfation site can mimic sclerostin binding and compete withfull length sclerostin for influencing Wnt signaling, as described inExample 12 below, establishes that post-translational sulfationmodifications of any protein subject to sulfation could influence theactivity of the protein, and that peptide fragments from the proteincomprising a sulfation site could be active.

The ability of the sclerostin peptide fragments to inhibitsclerostin-LRP binding was unexpected, particularly since thosefragments, comprising Y43 and Y213 are from a region of sclerostin thatwould not be expected to affect sclerostin-LRP binding. To illustratethis point, FIG. 9 is provided. That figure shows the sclerostin aminoacid sequence at the top, where Y43 and Y213 are underlined. The bottomshows an illustration, taken from Veverka et al., 2009, J. Biol. Chem.284:10890-10900, of three NMR-derived three-dimensional structures ofsclerostin. Panel A shows a best-fit superposition of 36 convergedstructures, Panel B shows a ribbon topology and Panel C shows aschematic representation of human sclerostin. As shown in Panel A, theN-terminal region (including Y43) and the C-terminal region (includingY213) are free-floating. As such, those regions would not be expected tointeract with LRP to influence sclerostin-LRP binding. However, as shownin Examples 12 and 13, 16-mer peptides comprising Y43 and Y213 were ableto reduce sclerostin binding to LRP, as well as reduce sclerostininhibition of Wnt signaling. This surprising result indicates thatsulfation sites, even when free-floating, can contribute to binding of aprotein ligand to its binding partner, and a peptide fragment of anysuch ligand comprising a sulfation site of that ligand would likelyinhibit binding of that ligand to its binding partner. In this regard,Weidauer et al. (2009, BBRC 380:160-165) has shown that removal of thefree-floating N-terminus and C-terminus affects the ability ofsclerostin to inhibit Wnt. However, that reference provides no insightinto the relevance of the N-terminus or C-terminus to sclerostin-LRPbinding. Furthermore, that reference does not provide any teaching orsuggestion that small peptides derived from any portion of sclerostinwould have an effect on the binding or activity of full lengthsclerostin.

The discovery that the N-terminus and C-terminus having sulfation sitesaffect the ability of sclerostin to bind to LRP and inhibit Wnt providesevidence that the sclerostin-LRP interaction involves interaction by 3domains—the binding region in the central portion of the molecule aswell as the interacting regions at each terminus. Each of these multipledomains of interaction is potentially a therapeutic or diagnostictarget. Further, the discovery that sulfation sites contribute to theprovision of interacting domains in the sclerostin-LRP ligand-bindingpartner interaction indicates that such multiple interactions likelyoccur in other ligand-binding partner interactions involving proteinsthat have sulfation sites.

Thus, the present invention is also directed to a composition comprisinga peptide comprising less than about 75 amino acids and/or amino acidanalogs including an amino acid or amino acid analog capable of beingpost-translationally sulfated. In these embodiments, the composition iscapable of inhibiting binding of a protein ligand comprising a sulfationsite to its binding partner. These embodiments can utilize any proteinligand now known or later discovered that has a sulfation site,including signal transduction proteins such as Wnt pathway proteins,immune proteins, or any other protein ligand.

In some of these embodiments, the peptide has at least about 50%homology to a fragment of the protein comprising the sulfation site. Insome aspects of these embodiments, the peptide has at least about 60%,70%, 80%, 90%, 95%, 98%, or 100% homology to a fragment of the proteincomprising the sulfation site. In other embodiments, the peptide is lessthan about 50, 30, 20, 15, 10, 8, 6 or 4 amino acids. In various aspectsof these embodiments, the sulfation site on the protein ligand is atyrosine. The peptide can be sulfated or unsulfated.

Nucleic acids comprising a nucleotide sequence encoding any of the abovepeptides is also provided, as are vectors (e.g., bacterial, yeast,mammalian, viral, expression, shuttle, plasmid, etc.) comprising thenucleic acid. Where the vector is an expression vector, the vector canfurther comprise control elements such that the peptide is expressedconstitutively or under the control of an inducible promoter.Prokaryotic and eukaryotic cells comprising these vectors are alsoenvisioned. These cells and vectors can be administered or implantedinto a mammal, e.g., a rodent or a human, e.g., for therapeuticpurposes.

Additionally, although the tyrosine modifications have been discussed interms of alterations of a protein's affinity for a binding partner in aheterodimeric interaction, dimerization is also an example of aprotein/protein interaction and as such, a homodimeric proteininteraction may also be influenced by sulfation modifications, and theprotein itself, should be included in the potential list of bindingpartners for the Wnt pathway protein. The degree of dimerization mayhave further effects with regard to binding to other proteins, where theaffinity of a dimeric protein may be higher than that of a monomericform. For instance, see Jekel et al., Biochimica Biophica Acta 19961291:195-198 where the affinity of a dimerized antigenic peptide ishigher than the monomeric form with regard to binding to an antibody. Inanother instance, TNF-α exists in trimeric form and binds to threereceptors simultaneously (Banner et al., 1993, Cell, 73:431-445). Sincedimerization or multimerization of proteins may be affected by sulfationprocesses, the methods above may also be applied to homodimericinteractions when the ability of a compound to affect sulfation is beinganalyzed. Assays that measure the ability of sulfated and unsulfatedprotein to form a complex with a binding partner may also be carried outwith another molecule of the protein as the intended binding partner.Antibodies may also be developed that are specific to dimers as comparedto monomers as previously described by Raven et al., in US PatentPublication 20050163776. Selectivity may be carried out by testing forthe ability to react with dimers and then counter-selecting byeliminating antibodies that exhibit cross-reactivity with the monomericform.

Another group of useful reagents provided herein are antibodies directedto the sulfation site. In the first place, the identification of thesulfation site offers evidence that the site is likely to be involved inprotein-protein interactions. Thus, for instance, the particular portionof the sclerostin protein involved in interaction with LRP5/6 has notbeen clearly identified, but the discovery of the sulfation site ofsclerostin in the amino terminal sequences described in Example 2provides a novel target for antibody binding that is likely to affectthe interaction of sclerostin with LRP5/6 that is different from thesclerostin sequences previously postulated by Ververka et al., 2009 JBC284:10,890-10,900, Weidauer et al., 2009 BBRC 380:160-165 and Krumlaufin U.S. Pat. No. 7,585,501.

Although the sulfonation modifications are not part of the sitesdescribed above by Ververka et al., 2009 and Weidauer et al. 2009, itshould be pointed out that their studies used sclerostin prepared fromE. coli and as such, they were studying sclerostin that lacked anypost-translational modifications. This offers the possibility ofincreased binding that augments the binding taking place through the“core” portion of sclerostin that they have studied. For instance, therecould be intra-strand interactions of the sulfonated tyrosines at thecarboxy or amino ends with the “core” portion of sclerostin that altersthe binding properties of the protein as a whole. Alternatively, bindingthrough the “core” portion can be augmented by a separate binding of themodified tyrosines of the sclerostin to its LRP5/6 binding partner. Ineither case, the overall effect is the further stabilization of theprotein/binding partner complex. Of course, it is understood that otherexplanations are also possible for the ability of the sulfonatedtyrosine(s) of sclerostin to enhance biding of sclerostin to LRRP5/6.This same rationale applies to other sulfated Wnt pathway proteins whenpotentially, sulfation can directly lead to an increased affinity in thecore region or by an intrastrand or interstrand binding event that leadsto an overall increase in complex stability.

Thus, provided is a protein comprising an antibody binding site thatbinds to a sulfated epitope of a Wnt pathway protein that is not Wnt5A,Wnt11, or Wnt3a. As used herein an “antibody binding site” is a portionof an immunoglobulin that binds to an antigen. The protein in theseembodiments will thus bind to a sulfated epitope of the Wnt pathwayprotein. The protein may also take any other form that is known in theart for use in immunodetection or immunotherapy.

The proteins of these embodiments include non-immunoglobulin proteinsfused, e.g., by chemical or recombinant DNA methods, to an antibodybinding site. In other embodiments, the protein is an antibody or anantibody fragment. For instance, the protein may be polyclonal,monoclonal, chimeric, human, humanized, bispecific, multispecific,primatized or an antibody fragment. Antibody fragments that me be of usein the present invention may comprise but not be limited to is Fab,ScFv, Fab′, F(ab′)₂, Fv, Fv(ab)₂ or aggregates thereof. The antibodybinding site may be derived from any vertebrate species including butnot limited to mice, rabbits, goats, sheep, chickens, camels, or humans.The antibody binding site can also be from any immunoglobulin classincluding IgG, IgM, or IgA.

In some embodiments, the antibody binding site can distinguish betweenthe sulfated and unsulfated sulfation site, e.g., where the antibodybinding site binds to the sulfated sulfation site but does notsubstantially bind to the unsulfated sulfation site. In otherembodiments, the antibody binding site does not distinguish between thesulfated and unsulfated forms, but are still specific for thesurrounding amino acids at the Wnt pathway protein sulfation site.

The antibody binding site can be directed against any Wnt pathwayprotein, for example a protein having a native sulfation site or aprotein that is engineered to have such a site. Nonlimiting examples ofproteins from which the peptide can be derived are a sclerostin, a Dvl1,a Dkk1, a Kr1, a Fz6, an LRP5, a Wnt, a Dvl2, a Dvl3, a Dkk2, a DKK3, aDKK4, a Kr2, a Fz1, a Fz2, a Fz3, a Fz4, a Fz5, a Fz7, a Fz8, a Fz9, aFz10, an LRP4, an LRP6 or any other Wnt pathway protein now known orlater discovered.

In one embodiment of the present invention, the epitope is five aminoacids or greater. In another embodiment, the epitope encompasses ten ormore amino acids. A comparison of the identity and location of potentialepitopes of the present invention and sequences used as sclerostinepitopes in prior art is given in FIG. 4. The underlined regionsadjacent to Tyr₄₃ and Tyr₂₁₃ in FIG. 4 are only intended to render avisual aid in comparing the regions of the present invention withpreviously described art and not intended to delineate the epitopeitself, or limit the epitopes to which the antibody binding site binds.Antibody binding sites may be generated that are specific for either thesulfated or unsulfated form of the protein and include the sulfationsite (e.g., Tyr₄₃ or Tyr₂₁₃ of human sclerostin, Tyr₈ of human Dvl1,Tyr₈₃ of human Dkk1, Tyr₁₇₅ or the Tyr₁₇₈ of human Kr1, Tyr₅₈₀ of humanFz6, or Tyr₃₈₀ or Tyr₁₅₈₃ of human LRP5 and non-human equivalentsthereof.

Although peptides may be used for the generation of linear epitopes,antibodies can also be found that recognize a three-dimensional set ofdeterminants (sometimes referred to as interrupted epitopes ornon-linear epitopes) and development and isolation of these types ofantibodies can be carried out by using three-dimensional antigens suchas the entire protein of interest or selected fragments as immunogens.Such antibodies may also be realized from screening of pre-formedlibraries that are independent of an immunogen. Screening can then becarried out for an ability to distinguish between sulfated andunsulfated versions of the protein of interest. For a discussion on theuse of conformationally derived epitopes, see Van Regenmortel 1998, JImmunol Methods 216:37-48; Villen et al., 2001 Biologicals 29:265-269;Moreau et al., 2006 Bioinformatics 22:1088-1095; and Huang and Honda2006 BMC Immunology 7:7.

The protein comprising an antibody binding site of these embodiments maybe prepared by any method known in the art including immunizationfollowed by bleeding or spleen cell fusion with myeloma cell lines, andrecombinant DNA technology including but not limited to phage displaytechnology. See also Bundgaard et al., 2008 Methods Mol Bio 446:47-66;Hoffhiner et al., 2006; Kehoe et al. 2006; Craig et al., 2009 Hybridoma28:377-381; U.S. Pat. No. 7,585,501, US Patent Publication 2004/0009535and US Patent Publication 2009/02130113, all of which are incorporatedby reference. One source of antigens that may be used for this purposeis artificial peptides that represent the sulfated sequences, forexample the peptides described above. The peptide or peptides used forimmunization may be modified or unmodified, depending upon whether theantibody is desired to recognize the modified or unmodified epitope.Post-synthetic modifications can be carried out either chemically or byin vitro modification by a sulfotransferase, for example by the methodsprovided in the Examples below. Screenings of antibody libraries canthen be carried out to determine the nature of the recognition such thatit is specific for the sulfated version of the target protein, theunsulfated form or is independent of the state of sulfation. In additionto such custom libraries, pre-existing libraries such as the HuCal phagelibrary is commercially available from AbD Serotec (Raleigh, N.C.) andis advertised as having more than 15 billion functional human antibodyspecificities. Another commercially available library comprises camelidderived antibodies and is available from Ablynx, Ghent, Belgium. Theselibraries have the advantage of not requiring any particular immunogenprior to screening. Screenings of this library may also be carried outas discussed above.

The presence of a sulfation group should in itself be sufficient todefine part of an epitope. In an analogous fashion for anotherpost-synthetic modification, the literature is replete with a largenumber of antibodies that are dependent on targets being either inphosphorylated or unphosphorylated forms and these form the basis ofnumerous assays for kinase activity. Furthermore, as describedpreviously, the presence or absence of such small chemical moieties as aphosphate or sulfate group can have profound effects upon activity, thusvalidating the ability of biological partners to be able to recognizethe differences between modified and unmodified forms. Specific examplesof the search and identification of antibodies that are specific toepitopes of target proteins comprising a sulfated tyrosine have beendescribed by Bundgaard et al. 2008, cited above. In a further example,an antibody (Mab15) that was selected for recognizing thyrotrophinreceptor (TSHr) was found to have an epitope that was only found inmature forms of its target protein suggesting that some form ofprocessing was required to create the appropriate epitope (Costagliolaet al., 2002 EMBO J 21:504-513). In vivo treatment of cells withchlorate (which as mentioned earlier reduces sulfation modifications)resulted in production of a mature protein that was now unrecognizableby Mab15 indicating that the antibody was able to distinguish betweenthe sulfated and unsulfated forms of its epitope and would only bind tothe sulfated version. Thus, even though it was not originally selectedfor this feature, the use of sulfated antigens allowed isolation andidentification of an antibody specific for a sulfate epitope in thistarget.

As discussed above, antibodies of this nature may also be used toevaluate in vitro assays of sulfation where they may be used to monitorconversion of the unsulfated form into the modified form. Theseantibodies may also be used alone or in conjunction with antibodies thatrecognize an epitope specific for the unsulfated form and/or forantibodies to an epitope in an amino acid sequence different from thesulfation sequence. Thus, for instance, an antibody that is specific forthe sulfated form of the protein may be used in conjunction with anantibody that is specific for an unsulfated region of the protein fornormalization purposes. In another example of use, an antibody that isspecific for the unsulfated form can be used in conjunction with anantibody that recognizes the same region but essentially offers nodiscrimination between the sulfated and unsulfated forms of the antigen.Alternatively, two antibodies can be used where one is specific for thesulfated form and another is for the unsulfated form.

The proteins comprising an antibody binding site described above areuseful for analytical or diagnostic purposes for evaluating the presenceof sulfated proteins and/or the extent of sulfation. As described above,shifts in sulfation levels have previously been noted to be a feature ofgastrin and cholcystokinin in cancer cells (Rehnfeld, 1990). The proteinsamples may be products that are excreted in the media or they may bederived from cell extracts. By these means, evaluation of physiologicallevels of sulfation of a Wnt pathway protein can be carried out withbiological specimens. These may be used in a variety of ways to comparespecimens that differ from each other in terms of origin, treatment orphysiological conditions. An antibody specific for a sulfated form of atarget protein may be used alone for this purpose or it may be combinedin an assay that further includes an antibody directed towards theunsulfated form or an antibody that recognizes both sulfated andunsulfated forms. In reference to the latter, an ability to recognizeboth sulfated and unsulfated forms may be a property of an antibody thatrecognizes the epitope where the sulfation is located but is genericallyindependent of the sulfation state, or it can an antibody that lacksrelevance to the sulfation state by recognizing an epitope that islocated outside of the modification region of the protein.

Thus, the present invention is also directed to a method of detecting orquantifying a sulfated Wnt pathway protein in a preparation. The methodcomprises combining the preparation with the protein comprising anantibody binding site described above under conditions allowing bindingof the protein comprising an antibody binding site to the sulfated Wntpathway protein in the preparation, then determining whether the proteincomprising an antibody binding site is specifically bound to thesulfated Wnt pathway protein in the preparation. Any of the proteincomprising an antibody binding site as described above can be used inthis method, including but not limited to antibodies or antibodyfragments.

These methods encompass the use of any immunological detection methoddescribed in the art, including immunoassays useful to detect the Wntpathway protein in an extract of a biological tissue, including but notlimited to ELISA, radioimmunoassay, and western blotting. Alsoencompassed within these methods are immunohistochemical methods for usein an intact tissue such as a fixed or unfixed tissue section. In someembodiments of these methods, the protein comprising an antibody bindingsite further comprises a detectable label, e.g., an enzyme, afluorescent moiety or an electron dense moiety, as they are known in theart.

The antibody binding site can be directed against any Wnt pathwayprotein, for example a protein having a native sulfation site or aprotein that is engineered to have such a site. Antibodies to Wntpathway proteins are well known. See, e.g., U.S. Patent Publication2006/0127393. Nonlimiting examples of proteins from which the peptidecan be derived are a sclerostin, a Dvl1, a Dkk1, a Kr1, a Fz6, an LRP5,a Wnt, a Dvl2, a Dvl3, a Dkk2, a DKK3, a DKK4, a Kr2, a Fz1, a Fz2, aFz3, a Fz4, a Fz5, a Fz7, a Fz8, a Fz9, a Fz10, an LRP4, an LRP6 or anyother Wnt pathway protein now known or later discovered.

The present invention also provides therapeutic methods using thecompositions described above for treatment of a variety of diseasesexacerbated by Wnt activation or inhibition. Nonlimiting examples ofdiseases exacerbated by Wnt activation include rheumatoid arthritis, acancer, anemia, immune deficiency, high bone mass, hyperparathyroidtumor, caudal duplication syndrome, tooth agenesis, familial adenomatouspolyposis, diabetic retinopathy, retinal inflammation, vascular leakage,and Wilms tumor. Nonlimiting examples of diseases exacerbated by Wntinhibition include osteoporosis, osteopenia, osteomalacia, osteogenesisimperfecta, avascular necrosis (osteonecrosis), poor healing ofimplants, bone loss due to other disorders, periodontal disease,osteoarthritis, arthritis, and the formation and/or presence ofosteolytic lesions, a cancer, type II diabetes, hair loss, inadequateproduction of stem cells, acute or chronic glomerulonephritis, rapidlyprogressive glomerulonephritis, nephrotic syndrome, focal proliferativeglomerulonephritis, systemic lupus erythematosus, Goodpasture'ssyndrome, polycystic kidney disease, acute tubular necrosis, acute renalfailure, polycystic renal disease, medullary sponge kidney, medullarycystic disease, nephrogenic diabetes, renal tubular acidosis, atubulointerstitial disease, acute and rapidly progressive renal failure,chronic renal failure, nephrolithiasis, gout, hypertension,nephrosclerosis, microangiopathic hemolytic anemia, atheroembolic renaldisease, diffuse cortical necrosis, renal infarcts, angina pectoris,myocardial infarction, chronic ischemic heart disease, hypertensiveheart disease, pulmonary heart disease, rheumatic fever, rheumatic heartdisease, endocarditis, mitral valve prolapse, aortic valve stenosis,valvular and vascular obstructive lesions, atrial or ventricular septaldefect, patent ductus arteriosus, myocarditis, congestivecardiomyopathy, hypertrophic cardiomyopathy, X-linked focal dermalhypoplasia, tetra-amelia, Mullerian-duct regression and viriliation,Fuhrmann syndrome, odonto-onchyo-dermal hypoplasia, obesity, XX sexreversal with palmoplanter hyperkeratosis, autosomal recessiveanonychia, hyponychia congenita, Van Buchem disease, or familialexudative vitreoretinopathy. See, e.g., MacDonald et al., 2009, Dev.Cell 17:9-26; Polakis, 2000, Genes Dev. 14:1837-1851; Chen et al., 2009,Am. J. Pathol. 175:2676-2685.

With respect to particular Wnt pathway proteins targeted in thesetherapeutic embodiments, sclerostin and Dkk1 (both antagonists of Wntsignaling) are particularly useful targets since those two proteins aresoluble proteins that interact with LRP5/6 in the intercellular space.Thus, administering either of these soluble proteins to a subject wouldbe expected to increase their Wnt-inhibiting effect (i.e., decrease Wntsignaling). Further, since sulfation increases the Wnt-inhibiting effectof these proteins, administration of the sulfated forms would beexpected to be more effective than administration of unsulfated or mixedsulfated and unsulfated forms. Conversely, administration of antibodiesto either protein to a subject would be expected to decrease theirWnt-inhibiting effect (i.e., increase Wnt signaling). Administration ofantibodies directed against Kr1 (also an antagonist of Wnt signaling)should also provide therapeutic value. Assays for binding or activity ofWnt pathway proteins such as Dkk1 or sclerostin are well known. See,e.g., Murrills et al., 2009, J. Cellular Biochem. 108:1066-1075.

Furthermore, with respect to sclerostin, although the binding ofsclerostin to an LRP receptor is responsible for biological effects, itis also known that sclerostin interacts with other proteins such as BMPs(Bone Morphogenic Proteins) (Winkler et al., 2003 EMBO J 22:6267-6276),Noggin (Winkler et al., 2004 J Biol Chem 279:36293-36298) and“Cysteine-rich protein 61” (Craig et al 2010 (BBRC 392:36-40). As such,the discovery of the sulfated amino acids in sclerostin allowsapplication of the present invention to interactions between sclerostinand these other proteins as well as the interactions with LRP receptors.

Thus, the invention is also directed to a method of inhibiting a Wntsignaling pathway. The method comprises contacting an LRP5/6 receptor inthe Wnt signaling pathway with any of the above-described compositionscomprising a sulfated peptide having homology to a sclerostin in amanner sufficient to inhibit the Wnt signaling pathway.

In some of these embodiments, the Wnt signaling pathway is in amammalian cell, for example a human cell. In aspects of theseembodiments, the mammalian cell is part of a mammal, e.g., a human. Inpreferred embodiments, the human has a disease exacerbated by Wntactivation. Non-limiting examples of the disease is rheumatoidarthritis, a cancer, anemia, immune deficiency, high bone mass,hyperparathyroid tumor, caudal duplication syndrome, tooth agenesis,familial adenomatous polyposis, diabetic retinopathy, retinalinflammation, vascular leakage, or Wilms tumor.

The invention is further directed to a method of enhancing a Wntsignaling pathway. The method comprises contacting an LRP5/6 receptor inthe Wnt signaling pathway with any of the above-described compositionscomprising a nonsulfated peptide having homology to a sclerostin in amanner sufficient to enhance the Wnt signaling pathway.

In some of these embodiments, the Wnt signaling pathway is in amammalian cell, for example a human cell. In aspects of theseembodiments, the mammalian cell is part of a mammal, e.g., a human. Inpreferred embodiments, the human has a disease exacerbated by Wntinhibition. Non-limiting examples of the disease is osteoporosis,osteopenia, osteomalacia, osteogenesis imperfecta, avascular necrosis(osteonecrosis), poor healing of implants, bone loss due to otherdisorders, periodontal disease, osteoarthritis, arthritis, and theformation and/or presence of osteolytic lesions, a cancer, type IIdiabetes, hair loss, inadequate production of stem cells, acute orchronic glomerulonephritis, rapidly progressive glomerulonephritis,nephrotic syndrome, focal proliferative glomerulonephritis, systemiclupus erythematosus, Goodpasture's syndrome, polycystic kidney disease,acute tubular necrosis, acute renal failure, polycystic renal disease,medullary sponge kidney, medullary cystic disease, nephrogenic diabetes,renal tubular acidosis, a tubulointerstitial disease, acute and rapidlyprogressive renal failure, chronic renal failure, nephrolithiasis, gout,hypertension, nephrosclerosis, microangiopathic hemolytic anemia,atheroembolic renal disease, diffuse cortical necrosis, renal infarcts,angina pectoris, myocardial infarction, chronic ischemic heart disease,hypertensive heart disease, pulmonary heart disease, rheumatic fever,rheumatic heart disease, endocarditis, mitral valve prolapse, aorticvalve stenosis, valvular and vascular obstructive lesions, atrial orventricular septal defect, patent ductus arteriosus, myocarditis,congestive cardiomyopathy, hypertrophic cardiomyopathy, X-linked focaldermal hypoplasia, tetra-amelia, Mullerian-duct regression andviriliation, Fuhrmann syndrome, odonto-onchyo-dermal hypoplasia,obesity, XX sex reversal with palmoplanter hyperkeratosis, autosomalrecessive anonychia, hyponychia congenita, Van Buchem disease, orfamilial exudative vitreoretinopathy. In some of these embodiments, thedisease is characterized by insufficient bone growth.

In further embodiments, the instant invention is directed to a method oftreating a subject having a disease exacerbated by Wnt activation. Themethod comprises obtaining a Wnt pathway protein that inhibits Wntactivation and comprises a sulfation site; treating the Wnt pathwayprotein with a sulfotransferase that causes sulfation of the Wnt pathwayprotein; and administering the treated Wnt pathway protein to thesubject. By treating the Wnt pathway protein with a sulfotransferaseprior to administration, complete sulfation of the protein is assured toprovide maximum Wnt inhibiting activity.

In preferred embodiments, the sulfated Wnt pathway protein isadministered in a pharmaceutically acceptable excipient. By“pharmaceutically acceptable” it is meant a material that (i) iscompatible with the other ingredients of the composition withoutrendering the composition unsuitable for its intended purpose, and (ii)is suitable for use with subjects as provided herein without undueadverse side effects (such as toxicity, irritation, and allergicresponse). Side effects are “undue” when their risk outweighs thebenefit provided by the composition. Non-limiting examples ofpharmaceutically acceptable carriers include, without limitation, any ofthe standard pharmaceutical carriers such as phosphate buffered salinesolutions, water, emulsions such as oil/water emulsions, microemulsions,and the like.

Any of the above-described proteins, peptides or compositions can beformulated without undue experimentation in pharmaceutically acceptableexcipient(s) for administration to a mammal, including humans, asappropriate for the particular application. Additionally, proper dosagesof the compositions can be determined without undue experimentationusing standard dose-response protocols.

Accordingly, the compositions designed for oral, lingual, sublingual,buccal and intrabuccal administration can be made without undueexperimentation by means well known in the art, for example with aninert diluent or with an edible carrier. The compositions may beenclosed in gelatin capsules or compressed into tablets. For the purposeof oral therapeutic administration, the pharmaceutical compositions ofthe present invention may be incorporated with excipients and used inthe form of tablets, troches, capsules, elixirs, suspensions, syrups,wafers, chewing gums and the like.

Tablets, pills, capsules, troches and the like may also contain binders,recipients, disintegrating agent, lubricants, sweetening agents, andflavoring agents. Some examples of binders include microcrystallinecellulose, gum tragacanth or gelatin. Examples of excipients includestarch or lactose. Some examples of disintegrating agents includealginic acid, cornstarch and the like. Examples of lubricants includemagnesium stearate or potassium stearate. An example of a glidant iscolloidal silicon dioxide. Some examples of sweetening agents includesucrose, saccharin and the like. Examples of flavoring agents includepeppermint, methyl salicylate, orange flavoring and the like. Materialsused in preparing these various compositions should be pharmaceuticallypure and nontoxic in the amounts used.

The compounds can easily be administered parenterally such as forexample, by intravenous, intramuscular, intrathecal or subcutaneousinjection. Parenteral administration can be accomplished byincorporating the compounds into a solution or suspension. Suchsolutions or suspensions may also include sterile diluents such as waterfor injection, saline solution, fixed oils, polyethylene glycols,glycerine, propylene glycol or other synthetic solvents. Parenteralformulations may also include antibacterial agents such as for example,benzyl alcohol or methyl parabens, antioxidants such as for example,ascorbic acid or sodium bisulfite and chelating agents such as EDTA.Buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose may also beadded. The parenteral preparation can be enclosed in ampules, disposablesyringes or multiple dose vials made of glass or plastic.

Rectal administration includes administering the compound, in apharmaceutical composition, into the rectum or large intestine. This canbe accomplished using suppositories or enemas. Suppository formulationscan easily be made by methods known in the art. For example, suppositoryformulations can be prepared by heating glycerin to about 120° C.,dissolving the composition in the glycerin, mixing the heated glycerinafter which purified water may be added, and pouring the hot mixtureinto a suppository mold.

Transdermal administration includes percutaneous absorption of thecomposition through the skin. Transdermal formulations include patches(such as the well-known nicotine patch), ointments, creams, gels, salvesand the like.

The present invention includes nasally administering to the subject atherapeutically effective amount of the compound. As used herein,nasally administering or nasal administration includes administering thecompound to the mucous membranes of the nasal passage or nasal cavity ofthe patient. As used herein, pharmaceutical compositions for nasaladministration of the composition include therapeutically effectiveamounts of the protein prepared by well-known methods to beadministered, for example, as a nasal spray, nasal drop, suspension,gel, ointment, cream or powder. Administration of the protein may alsotake place using a nasal tampon or nasal sponge.

Where the composition is administered peripherally such that it mustcross the blood-brain barrier, the composition is preferably formulatedin a pharmaceutical composition that enhances the ability of thecompound to cross the blood-brain barrier of the mammal. Suchformulations are known in the art and include lipophilic compounds topromote absorption. Uptake of non-lipophilic compounds can be enhancedby combination with a lipophilic substance. Lipophilic substances thatcan enhance delivery of the compound across the nasal mucus include butare not limited to fatty acids (e.g., palmitic acid), gangliosides(e.g., GM-1), phospholipids (e.g., phosphatidylserine), and emulsifiers(e.g., polysorbate 80), bile salts such as sodium deoxycholate, anddetergent-like substances including, for example, polysorbate 80 such asTween™, octoxynol such as Triton™ X-100, and sodiumtauro-24,25-dihydrofusidate (STDHF). See Lee et al., Biopharm., April1988:3037.

In particular embodiments of the invention, the protein is combined withmicelles comprised of lipophilic substances. Such micelles can modifythe permeability of the nasal membrane to enhance absorption of theprotein. Suitable lipophilic micelles include without limitationgangliosides (e.g., GM-1 ganglioside), and phospholipids (e.g.,phosphatidylserine). Bile salts and their derivatives and detergent-likesubstances can also be included in the micelle formulation. The proteincan be combined with one or several types of micelles, and can furtherbe contained within the micelles or associated with their surface.

Alternatively, the protein can be combined with liposomes (lipidvesicles) to enhance absorption. The protein can be contained ordissolved within the liposome and/or associated with its surface.Suitable liposomes include phospholipids (e.g., phosphatidylserine)and/or gangliosides (e.g., GM-1). For methods to make phospholipidvesicles, see for example, U.S. Pat. No. 4,921,706 to Roberts et al.,and U.S. Pat. No. 4,895,452 to Yiournas et al. Bile salts and theirderivatives and detergent-like substances can also be included in theliposome formulation.

In various embodiments, the Wnt pathway protein is administeredparenterally, e.g., intravenously.

The Wnt pathway protein can be treated with the sulfotransferase by anymethod known in the art. In some embodiments, the Wnt pathway protein istreated with the sulfotransferase 117 vitro. In other embodiments, theWnt pathway protein is produced in a cell that further comprises thesulfotransferase. The cell in these embodiments can be a eukaryotic celland/or a cell that expresses a recombinant sulfotransferase. Thesulfotransferase in these embodiments is preferably a TPST1 or TPST2.The Wnt pathway protein can also be sulfated by chemical methods.

The Wnt pathway protein in these embodiments can be any inhibitory Wntpathway protein that comprises a sulfation site, whether the sulfationsite is native or not native to the protein. In preferred embodiments,the Wnt pathway protein is a sclerostin or a Dkk1.

These methods are useful for the treatment of any disease, now known orlater discovered, that is exacerbated by Wnt activation, including butnot limited to rheumatoid arthritis, a cancer, anemia, immunedeficiency, high bone mass, hyperparathyroid tumor, caudal duplicationsyndrome, tooth agenesis, familial adenomatous polyposis, diabeticretinopathy, retinal inflammation, vascular leakage, or Wilms tumor.

In other embodiments, the invention is directed to another method oftreating a subject having a disease exacerbated by Wnt activation. Thismethod comprises (a) obtaining the composition comprising a peptideconsisting of a fragment of a Wnt pathway protein that is not the entireWnt pathway protein, where the fragment is sulfated, as described above,and (b) administering the composition to the subject. As discussedabove, the peptide can comprise amino acid analogs and can furthercomprise some amino acid changes from the native Wnt pathway protein.

In these embodiments, the Wnt pathway protein inhibits Wnt activation.Administration of such a peptide would be expected to effectivelyinhibit Wnt signaling. Preferably, the composition is formulated in apharmaceutically acceptable excipient, as described above.

In various embodiments, the peptide is less than 75 amino acids long, asdescribed above.

The peptide can be administered by any means known in the art, asdescribed in the above discussion of pharmaceutically acceptableexcipients. In some embodiments, the peptide is administeredparenterally, e.g., intravenously.

The peptide can be a fragment of any inhibitory Wnt pathway protein thatcomprises a sulfation site, whether the sulfation site is native or notnative to the protein. In preferred embodiments, the Wnt pathway proteinis a sclerostin or a Dkk1.

The peptide can be sulfated by any means known in the art, e.g., bytreatment with a sulfotransferase in vitro or by producing the peptidein a cell that further comprises the sulfotransferase. The cell in theseembodiments can be a eukaryotic cell and/or a cell that expresses arecombinant sulfotransferase. The sulfotransferase in these embodimentsis preferably a TPST1 or TPST2.

These methods are useful for the treatment of any disease, now known orlater discovered, that is exacerbated by Wnt activation, including butnot limited to rheumatoid arthritis, a cancer, anemia, immunedeficiency, high bone mass, hyperparathyroid tumor, caudal duplicationsyndrome, tooth agenesis, familial adenomatous polyposis, diabeticretinopathy, retinal inflammation, vascular leakage, or Wilms tumor.

The proteins comprising an antibody binding site that binds to asulfated Wnt pathway protein, as described above, may find use astherapeutic reagents that disrupt interaction between the Wnt pathwayprotein and its binding partner, for example the binding of sclerostinor Dkk1 to LRP5/6. In the case of antibodies that are specific foreither sulfated or unsulfated forms, a finer degree of control can beexerted over physiological processes, since each type of antibody willbe directed towards a subpopulation of the target protein. As such, anability to target only the sulfated form will leave the activity of theunsulfated from intact and vice versa for an antibody to the unsulfatedfrom. This is a level of discrimination that would not be produced byantibodies described previously for Wnt pathway proteins. On the otherhand, an antibody of the present invention that is generic in the senseof being independent of the sulfation state of the Wnt pathway protein,may also have therapeutic utility because the sites where modificationstake place may have more significance than previously recognized, andthus, these regions are novel epitopes that are useful as targets forimmunotherapy.

Thus, the present invention is also directed to a method of treating asubject having a disease exacerbated by Wnt inhibition. The methodcomprises treating the subject with the protein comprising an antibodybinding site to a sulfated epitope of a Wnt pathway protein as describedabove. In these embodiments, the Wnt pathway protein enhances Wntinhibition. In some embodiments, the protein is an antibody or anantibody fragment.

The inhibitory Wnt pathway protein to which the antibody binding site isdirected can be any such protein now known or later discovered, wherethe sulfated epitope is either native or engineered into the protein. Inpreferred embodiments, the Wnt pathway protein is a sclerostin, a Dkk1,or a Kr1.

The protein comprising an antibody binding site can be administered byany means known in the art as described in the above discussion ofpharmaceutically acceptable excipients. In some embodiments, the proteinis administered parenterally, e.g., intravenously.

These methods are useful for the treatment of any disease, now known orlater discovered, that is exacerbated by Wnt inhibition, including butnot limited to osteoporosis, osteopenia, osteomalacia, osteogenesisimperfecta, avascular necrosis (osteonecrosis), poor healing ofimplants, bone loss due to other disorders, periodontal disease,osteoarthritis, arthritis, and the formation and/or presence ofosteolytic lesions, a cancer, type II diabetes, hair loss, inadequateproduction of stem cells, acute or chronic glomerulonephritis, rapidlyprogressive glomerulonephritis, nephrotic syndrome, focal proliferativeglomerulonephritis, systemic lupus erythematosus, Goodpasture'ssyndrome, polycystic kidney disease, acute tubular necrosis, acute renalfailure, polycystic renal disease, medullary sponge kidney, medullarycystic disease, nephrogenic diabetes, renal tubular acidosis, atubulointerstitial disease, acute and rapidly progressive renal failure,chronic renal failure, nephrolithiasis, gout, hypertension,nephrosclerosis, microangiopathic hemolytic anemia, atheroembolic renaldisease, diffuse cortical necrosis, renal infarcts, angina pectoris,myocardial infarction, chronic ischemic heart disease, hypertensiveheart disease, pulmonary heart disease, rheumatic fever, rheumatic heartdisease, endocarditis, mitral valve prolapse, aortic valve stenosis,valvular and vascular obstructive lesions, atrial or ventricular septaldefect, patent ductus arteriosus, myocarditis, congestivecardiomyopathy, hypertrophic cardiomyopathy, X-linked focal dermalhypoplasia, tetra-amelia, Mullerian-duct regression and viriliation,Fuhrmann syndrome, odonto-onchyo-dermal hypoplasia, obesity, XX sexreversal with palmoplanter hyperkeratosis, autosomal recessiveanonychia, hyponychia congenita, Van Buchem disease, or familialexudative vitreoretinopathy.

Also provided herein is another method of treating a subject having adisease exacerbated by activation of a Wnt signaling pathway. The methodcomprises administering the above-described composition comprising asulfated peptide homologous to a sclerostin to the subject in a mannersufficient to inhibit the Wnt signaling pathway. In some of theseembodiments, the composition is administered to a tissue of the subjectthat is affected by the disease, in order to more specifically directthe treatment to the disease.

In various embodiments, the disease is rheumatoid arthritis, a cancer,anemia, immune deficiency, high bone mass, hyperparathyroid tumor,caudal duplication syndrome, tooth agenesis, familial adenomatouspolyposis, diabetic retinopathy, retinal inflammation, vascular leakage,or Wilms tumor.

In related embodiments, the present invention provides an additionalmethod of treating a subject having a disease exacerbated by inhibitionof a Wnt signaling pathway. The method comprises administering theabove-described composition comprising a nonsulfated peptide homologousto a sclerostin to the subject in a manner sufficient to reduce theinhibition of the Wnt signaling pathway. In some of these embodiments,the composition is administered to a tissue of the subject that isaffected by the disease, in order to more specifically direct thetreatment to the disease.

In various embodiments, the disease is osteoporosis, osteopenia,osteomalacia, osteogenesis imperfecta, avascular necrosis(osteonecrosis), poor healing of implants, bone loss due to otherdisorders, periodontal disease, osteoarthritis, arthritis, and theformation and/or presence of osteolytic lesions, a cancer, type IIdiabetes, hair loss, inadequate production of stem cells, acute orchronic glomerulonephritis, rapidly progressive glomerulonephritis,nephrotic syndrome, focal proliferative glomerulonephritis, systemiclupus erythematosus, Goodpasture's syndrome, polycystic kidney disease,acute tubular necrosis, acute renal failure, polycystic renal disease,medullary sponge kidney, medullary cystic disease, nephrogenic diabetes,renal tubular acidosis, a tubulointerstitial disease, acute and rapidlyprogressive renal failure, chronic renal failure, nephrolithiasis, gout,hypertension, nephrosclerosis, microangiopathic hemolytic anemia,atheroembolic renal disease, diffuse cortical necrosis, renal infarcts,angina pectoris, myocardial infarction, chronic ischemic heart disease,hypertensive heart disease, pulmonary heart disease, rheumatic fever,rheumatic heart disease, endocarditis, mitral valve prolapse, aorticvalve stenosis, valvular and vascular obstructive lesions, atrial orventricular septal defect, patent ductus arteriosus, myocarditis,congestive cardiomyopathy, hypertrophic cardiomyopathy, X-linked focaldermal hypoplasia, tetra-amelia, Mullerian-duct regression andviriliation, Fuhrmann syndrome, odonto-onchyo-dermal hypoplasia,obesity, XX sex reversal with palmoplanter hyperkeratosis, autosomalrecessive anonychia, hyponychia congenita, Van Buchem disease, orfamilial exudative vitreoretinopathy.

According to a further embodiment of the invention, the proteins,peptides or antibodies administered as described above may be employedas adjunct or adjuvant to other therapy, e.g. a therapy using a boneresorption inhibitor, for example as in osteoporosis therapy, inparticular a therapy employing calcium, a calcitonin or an analogue orderivative thereof, e.g. salmon, eel or human calcitonin, calcilytics,calcimimetics (e.g., cinacalcet), a steroid hormone, e.g. an estrogen, apartial estrogen agonist or estrogen-gestagen combination, a SERM(Selective Estrogen Receptor Modulator) e.g. raloxifene, lasofoxifene,bazedoxifene, arzoxifene, FC1271, Tibolone (Livial®), a SARM (SelectiveAndrogen Receptor Modulator), a RANKL antibody (such as denosumab), acathepsin K inhibitor, vitamin D or an analogue thereof or PTH, a PTHfragment or a PTH derivative e.g. PTH (1-84) (such as Preos™), PTH(1-34) (such as Forteo™), PTH (1-36), PTH (1-38), PTH (1-31)NH2 or PTS893. According to another embodiment, the antibodies of the inventionmay be employed in combination with other current osteoporosis therapyapproaches, including bisphosphonates (e.g., Fosamax™ (alendronate),Actonel™ (risedronate sodium), Boniva™ (ibandronic acid), Zometa™(zoledronic acid), Aclasta™/Reclast™ (zoledronic acid), olpadronate,neridronate, skelid, bonefos), statins, anabolic steroids, lanthanum andstrontium salts, and sodium fluoride. When pharmacological agents ofantibodies of the present invention are administered together withanother agent, the two can be administered in either order (i.e.sequentially) or simultaneously.

More generally, the present invention is directed to a method ofinhibiting binding of a protein ligand comprising a sulfation site toits binding partner. The method comprises adding the compositiondescribed above that is capable of inhibiting binding of a proteinligand comprising a sulfation site to its binding partner to the proteinligand and its binding partner in a manner sufficient to inhibit bindingof the protein ligand to its binding partner. This method could be usedwith any protein ligand that comprises a sulfation site.

Preferred embodiments are described in the following examples. Otherembodiments within the scope of the claims herein will be apparent toone skilled in the art from consideration of the specification orpractice of the invention as disclosed herein. It is intended that thespecification, together with the examples, be considered exemplary only,with the scope and spirit of the invention being indicated by theclaims, which follow the examples.

Example 1 In Vitro Sulfation of Sclerostin

Human sclerostin (25 μg R&D Systems, Minneapolis, Minn.)) wasreconstituted in 100 of 100 mM MES pH=7.0. Sulfation was carried out bymixing 50 μl (12.5 μg) human sclerostin and 22.5 μl (10.0 μg) of humanTPST1 (R&D Systems) with 125 μL of assay mix [78.87 mM MES pH 7.0, 2.5mM MgCl₂, 2.5 mM MnCl₂ 1.25 mM CaCl₂ and 200 μM PAPS (Sigma)].Incubation was carried out for 1.5 hrs at 37° C. Buffer was thenexchanged into 10 mM Tris pH 7.5 using protein desalting spin columns(Pierce Biochemicals, Rockford, Ill.).

Example 2 Detection of Sulfation Modifications by MS Analysis

Peptides from the sclerostin from Example 1 as well as untreatedSclerostin were digested with either trypsin or GluC and loaded onto aC18 column followed by injection into a LTQ mass spectrometer. In thefirst analysis, the mass spectrometer was instructed to make MS/MS ofall eluting peptides. The resulting data was analyzed and three peptidesfrom sclerostin containing tyrosines were identified: LGEYPEPPPELE (SEQID NO:2), YVTDGPCR (SEQ ID NO:3) and ANQAELENAY (SEQ ID NO:4). In thesecond analysis, targeted analysis was performed where the massspectrometer was instructed to only do MS/MS on masses corresponding tothe putative sulfated tyrosine containing peptides. For the GluC sample,the mass spectrometer was set to perform MS/MS at m/z 725.6, the mass ofthe doubly charged peptide LGEYPEPPPELE (SEQ ID NO:2) plus sulfation, ata normalized collision energy for CID at 2%, 4% or 10% and an MS3 of thehighest fragment in each of the three MS/MS. In the tryptic sample,MS/MS was performed at m/z 602.0, the mass of the doubly charged peptideANQAELENAY (SEQ ID NO:4) plus sulfation, and at m/z 524.5, the mass ofthe doubly charged peptide YVTDGPCR (SEQ ID NO:3) plus sulfation, at anormalized collusion energy for CID at 2%, 4% and 10% and an MS3 of thehighest fragment in each of the three MS/MS. Both peptides at m/z 725.6and 602.0, corresponding to sulfated peptide LGEYPEPPPELE (SEQ ID NO:2)and ANQAELENAY (SEQ ID NO:4), respectively, showed a neutral loss of 80Da (40 Da for a 2+ ion) at 10% CE which suggests that these peptideswere sulfated, whereas at 2% and 4%, the loss was not very pronounced(FIG. 1). The neutral loss fragments were subsequently fragmented andproduced the expected MS/MS for the expected peptides. The MS/MS at m/z524.5 did not show such a loss (data not shown). Essentially the sameresults were seen for both the untreated sclerostin and the sclerostinfrom Example 1 indicating the presence of sulfation modifications inSclerostin prior to the in vitro reaction with TPST-1 in Example 1.Furthermore, although a phosphate addition at this site would alsoresult in a shift of ˜80 kd higher weight, further tests showed that themodifications at these sites exhibited the chemical lability typical ofa sulfation modification.

Example 3 Biological Effects of In Vitro Sulfation of Sclerostin

A) Effects of Sulfation on Binding of Sclerostin to LRP5

1) Preparation of Alkaline Phosphatase-Labeled LRP5 (AlkPhos-LRP5)

293T cells were seeded into 9 cm dishes. The next day, each dish wastransfected with 12) 4 of LRP5R1/2AP construct using Lipofactamine Plus(Invitrogen, Carlsbad, Calif.) according to the manufacturer'sinstructions. LRP5R1/2-AP is a nucleic acid construct that expressesLRP5 extracellular domains 1 and 2 fused to alkaline phosphatase. 48hours after transfection, the supernatant of the culture was collectedas LRP5R1/2AP conditioned medium and concentrated 20 times using aCentricon unit (Millipore, Billerica, Mass.) and stored at −80° C.

2) Binding of AlkPhos-LRP5 to Sclerostin

Various amounts of unmodified sclerostin or the in vitro treatedsclerostin from Example 1 were diluted into 80 μl of TBST buffer andadded to individual wells of 96 well plates. After overnight incubation,unbound proteins were removed after which point the coated plates wereblocked with 3% nonfat milk in PBS. The plates were than decanted and0.5×LRP5R1/2AP conditioned medium was added to the plates. After 2.5hours, the conditioned medium was removed and the 96 well plates werewashed five times for three minutes with TBST. The alkaline phosphataseactivity in each well was then determined using the Tropix luminescenceassay kit (Invitrogen, Carlsbad, Calif.).

3) Results of the Binding Assay

As seen in FIG. 2, the sclerostin treated in vitro with TPST showed amarked increase in the amount of AlkPhos-LRP5 bound to the plates whencompared to the untreated sclerostin. These results are best interpretedas evidence that there is an increase in the binding affinity of thetreated protein compared to the starting material. These comparativeresults were repeated with the mouse versions of sclerostin (not shown)and showed essentially similar results although the basal levels of theproteins were different for each source.

B) Effects of Sulfation on the Ability of Sclerostin to Block WntInduced Expression of Alkaline Phosphatase

1) Induction of Alkaline Phosphatase Activity

Growing cultures of 10T1/2 cells were washed with PBS and trypsinizedfor 5 minutes. Cells were resuspended at a concentration of 6×10⁵cells/ml and 10 μl were seeded into individual wells of a Costar 96 wellplate (Corning, Inc.). Wnt 3a and either the untreated sclerostin or thesclerostin from Example 1 were added and the plates were incubated at37° C. for 24 hours. 50 μl of universal lysis buffer (from theLuciferase Reporter Gene Assay, Roche Applied Science, Indianapolis,Ind.) was added to each well at ambient temperature for 5 minutes.Detection of alkaline phosphatase was measured by the addition of 50 μlof ready-to-use CPSD with Sapphire Enhancer (Applied Biosystems)followed by an incubation at ambient temperature for 25 minutes.

2) Results of the Assay

As seen in FIG. 3, the sclerostin sulfated in Example 1 gave similarresults compared to the untreated sclerostin except at the highest levelof sclerostin input where there was a significantly (P=0.006) moreefficient blockage of Wnt induced alkaline phosphate activity. Thisresult for the highest level of sclerostin may be a result of theincrease in the binding affinity of the treated protein compared to thestarting material as seen in FIG. 2.

It should be noted that the sclerostin used in these experiments wasderived from recombinant clones in eukaryotic cell lines. Consequently,as seen in the MS results in Example 2, there is a significantpopulation of sclerostin proteins that already have pre-existingsulfation modifications. Thus, the positive effects seen in theexperiments above is the result of conversion of any remainingunsulfated forms into the sulfated version by TSPT-1.

Example 4 Evaluation of Sclerostin Sequences with “Sulfinator” Program

The “Sulfinator” program is an online methodology of predicting thepresence of sites in proteins that are substrates for tyrosine sulfation(Monigatti et al. 2002 Bioinformatics 18; 769-770). It can be accessedat the website having the http: address expasy.org/tools/sulfinator/with documentation available at the http: addressexpasy.org/tools/sulfinator/sulfinator-doc.html. When this program wasapplied to the human sclerostin sequence (UniProtKB Accession No.Q9BQB4), the amino acid sequence ELGEYPEPPPELENNK (SEQ ID NO:5) in the Nterminal region of sclerostin was identified as corresponding to atyrosine sulfation site with sulfation taking place with Tyr₄₃ inagreement with the MS results from Example 2. The correspondingsequences in the mouse and rat are GLGEYPEPPPENNQTM (SEQ ID NO:6) andGLREYPEPPQELENNQ (SEQ ID NO:7) respectively (UniProtKB Accession NoQ99P68 and Q99P67) where differences in the amino sequence areunderlined. Evaluation of the mouse and rat sclerostin sequences by theSulfinator program revealed that the rat protein should also be sulfated(and at the corresponding Tyr residue) while the mouse sequence did notshow a positive result. It should be noted, however, that part of thecriteria used by the Sulfinator program is contextual neighboring aminoacid sequences and when the oligopeptide GLGEYPEPPPENNQTM (SEQ ID NO:6)from the mouse sclerostin sequences was independently tested, it wasindicated as being potential site for sulfation. The loose structure atthe amino terminal end of Sclerostin (to be discussed below) is likelyresponsible for the oligopeptide Sulfinator results of mouse Sclerostinbeing in agreement with the binding assay results.

The region of sclerostin involved in binding to LRP5/6 is not preciselyknown. It has been described as “Finger 2” (˜aa's 115-147) by Weidaueret al., (2009 BBRC 380:160-165) and “Loop 2” (˜aa's 86-112) by Veverkaet al., (2009 JBC 284:10,890-10,900) where amino acid assignments arebased on the mature protein. It can be seen that neither putativelocation corresponds to the Tyr₄₃ sulfation site. Nonetheless, avisualization of the predicted 3-dimensional structure shows that Tyr₄₃is part of a loosely organized peptide strand that could located inproximity with the binding site in “loop 2” predicted by Ververka et al.As such, it is possible that the amino terminal portion of sclerostinalso participates in binding of sclerostin to LRP5/6 and sulfation mayhave effects on this particular protein/protein interaction. Furthersupport is from U.S. Pat. No. 7,585,501 where the Tyr₄₃ site is a shortdistance away from an additional sclerostin sequence (#15) that wasdescribed as participating in binding with LRP5/6. This point isillustrated further in FIG. 4.

Example 5 Peptides Derived from Sulfation Sites

Peptides from the sulfation modification sites regions may be useful inmodulating protein-protein interactions between a sulfated protein and abinding partner. Thus, for example, the sequences ELGEYPEPPPELENNK (SEQID NO:5) and KANQAELENAY (SEQ ID NO:8) from sclerostin can be used toartificially synthesize peptides that can be used as therapeuticcompounds. Both modified and unmodified versions of these peptides canbe made and tested to see which ones are more effective and if they areequivalent in potency.

Example 6 Development of Antibodies Specific for Sulfated Proteins

Antibodies that are specific for sclerostin can be developed usingpeptides derived from the recognition sequences described in Examples 2and 5. In FIG. 4, the sites previously described for use as epitopes forsclerostin antibodies is compared with the sulfation sites described inExample 2. Unmodified peptides can be designed and obtained fromnumerous commercial sources. Post-synthetic modifications can then becarried out either chemically or by in vitro modification by TPST-1.These antigens can then be used to obtain antibodies using methodstaught in Bundgaard et al., 2008; Hoffhiner et al., 2006; Kehoe et al.2006; U.S. Pat. No. 7,585,501: US Patent Publication 2004/0009535; andUS Patent Publication 2009/02130113. Screenings can be carried out todetermine the nature of the recognition such that it is specific forsulfation of only the target protein. A similar program can be carriedout with analogous peptides that remain unmodified; these can be used toobtain antibodies that are specific for the unmodified version of thetargets. Screenings can also be based upon an ability to bind to thespecific region of the sclerostin sulfation, but the affinity of theprotein is for both sulfated and unsulfated versions of the antigentarget.

The discovery of a sequence in sclerostin that comprises a sulfatemodified tyrosine provides information concerning previously unknownepitopes in sclerostin that may be used to generate novel antibodiesthat target these sites. For this purpose, a peptide can be used thatcomprises the sequence ELGEYPEPPPELE (SEQ ID NO:9) where the tyrosine ismodified to comprise a sulfate group in order to generate an antibodythat targets the sulfated tyrosine site at the amino end of sclerostin.This modification can be carried out either chemically or by treatmentwith TPST-1 and PAPS. Another peptide, comprising the sequenceKANQAELENAY (SEQ ID NO:10) (where the tyrosine is also modified bysulfation) can be used to generate an antibody to the sulfated tyrosinesite at the carboxyl end of sclerostin. Generation and isolation of anantibody can then be carried out by the methods described by Bundgaardet al., 2008 in conjunction with the methods taught in U.S. Pat. No.7,585,501, US Patent Publication 20040009535 and US Patent Publication20090130113, all of which are incorporated by reference.

When using a peptide with a sulfated tyrosine as the immunogen,resultant antibodies can display a variety of different affinities. Forexample, in an article giving the protocol for generating antibodiesagainst peptides containing a phosphorylated tyrosine, the point ismade: “Such an immunization will generate an immune response with atleast four components: (1) anti-carrier protein reactivity, (2) generalantiphosphotyrosine reactivity, (3) phosphorylation-independentanti-peptide reactivity and (4) phosphorylation-dependent anti-peptidereactivity.” (DiGiovanna et al., 2002 Current Protocols in Cell Biology16.6.1-16.6.18). As such, that article points out that even when using apeptide with the appropriate modification, antibodies can be generatedthat may only require the appropriate amino acid sequence and ignore thepresence or absence of a modified tyrosine. Consequently, many of thepast efforts to isolate an antibody against a phosphorylated peptidehave included a counter-selection step to eliminate antibodies that bindto the unphosphorylated version of the target peptide/protein.

In contrast, although it is a goal of the present invention to generateand isolate antibodies that are specific for a protein that has asulfated tyrosine, utility is also found during such a search toidentify and isolate antibodies that are specific for the sulfatedtyrosine site but that are also independent of the sulfation state ofthe target protein. Thus in parallel, identification processes can becarried out that initially are identified in terms of an the ability tobind to the region encompassed by the sulfation modifications and then asecondary screening can be carried out for a) antibodies that have theability to detect only epitopes that include the sulfation modificationand b) antibodies that are independent of the sulfation status of thetarget region.

Example 7 Growth of Sclerostin in Cells Treated with Chlorate andSubsequent Testing in Binding Assay

1. Production of Human Sclerostin

After amplification with the forward primer CAGGGGTGGCAGGCGTTCAA (SEQ IDNO:11) and the reverse primer GTAGGCGTTCTCCAGCTCGG (SEQ ID NO:12), thehuman sclerostin PCR product with blunt ends was cloned intopFastBac/HEM-TOPO vector (Invitrogen). The recombinant plasmid wastransformed into the DH10Bac™ E. coli strain. A transpositionsubsequently took place between the mini-Tn7 element on the pFastBac/HBMrecombinant plasmid and the mini-attTn7 target site on the baculovirusshuttle vector in the presence of transposition proteins from the helperplasmid, generating a recombinant bacmid. The recombinant bacmid DNA wasprepared and transfected into the Sf9 insect cell line with theCellfection II™ reagent (Invitrogen), from which the recombinantbaculovirus expressing human Sclerostin was produced. Human sclerostinwas produced in High Five™ insect cells (Invitrogen) infected with therecombinant baculovirus.

2. Preparation of Sclerostin in Insect Cells Treated with Chlorate andSubsequent Testing in Binding Assay

A. Preparation of Sclerostin in Insect Cells Treated with Chlorate

The SF9 cells described above were harvested, counted and diluted bySf-900 II SFM growth medium. The cells were distributed into 15 cmtissue culture dishes to reach 70-80% confluence and cultured for 6hours. The baculovirus expressing sclerostin was added into the cells at0.2 MOI. Infected Sf9 cells were cultured in the dishes at 28° C. for 72hrs. Culture medium was collected, centrifuged at 1200 rpm for 25minutes, and the supernatant was transferred into 50 ml tubes and keptat 4° C. A plaque assay was performed to determine the titer of thesclerostin-expressing baculovirus which was adjusted to ˜10⁷/ml withExpress Five™ Serum Free Medium. High Five™ cells were harvested,counted and diluted into Express Five™ Serum Free Medium (Invitrogen).Sclerostin-expressing baculovirus was added to the High Five™ cells at 2MOI, and the cell density was adjusted to 10⁶/ml in Express Five™ SerumFree Medium. After mixing, the cells were divided into two treatments.To one treatment, chlorate was added to a final concentration of 1 mM.The other treatment lacked chlorate. Each cell suspension was aliquotedinto eight 250 ml flasks, 100 ml/flask. The flasks were placed in ashaker at 28° C. and 100 rpm for 48 hr. The cell suspension was thencollected by centrifugation at 1500 rpm for 20 minutes and pooled. Atotal of 800 ml of supernatant was collected and frozen at −80° C.Elution Buffer A (25 mM imidazole in PBS+0.5% CHAPs) and B (400 mMimidazole in PBS+0.5% CHAPs) were prepared 50 ml Elution Buffer A wascombined with 7. 5 ml Ni-NTA agarose and the mixture was loaded onto acolumn. 800 ml of frozen conditioned medium was thawed. CHAPS (4 g) wasadded to the supernatant to reach a concentration of 0.5% CHAPS. Thecollected supernatant was loaded onto the Ni-NTA agarose column andeluted at ˜2 ml/min. After the supernatant was completely loaded ontothe column, the column was washed with 100 ml Buffer A at 2 ml/min towash out the unbound proteins. After washing, sclerostin was eluted fromthe column with 10 ml Buffer B. The sclerostin solution was loaded on toa centrifugal filter unit, after which PBS+0.5% CHAPS was added to makethe total volume 15 ml in the filter unit. The filter unit was thencentrifuged at 3000 rpm for 10 min. The centrifugation step was repeatedthree times. The sclerostin solution in the centrifugal filter unit wascollected and dried by lyophilization.

B. Sclerostin Binding Assay

Stock solutions of 0.1 μg “Normal” (sulfated) and “Unmodified” (chloratetreated) sclerostin/ml in PBS was prepared. 50 μl of the stock solutionwas diluted into 4000 μl PBS and 40 μl was loaded into each well of amicrotiter plate to coat the plate with sclerostin. The assay was thencarried out essentially as described in Example 3(A).

C. Results

Results are shown in FIG. 5. As shown therein, the “Normal” sulfatedsclerostin bound more LRP5 than the “Unmodified” chlorate treatedsclerostin.

Example 8 Reversal of Properties of Sclerostin Derived from ChlorateTreated Cells by Carrying Out In Vitro Sulfonation with TPST-1

MES buffer (0.1 M MES, 0.5% Triton X100, 2.5 mM MgCl₂, 2.5 mM MnCl₂,1.25 mM CaCl₂, 0.75 mg/mL BSA, pH 7.0) was prepared. PAPS(3′-phosphoadenosine-5′-phosphosulfate—the sulfate donor in the TPSTsulfation reaction) and TPST1 were dissolved in MES buffer to aconcentration of 10 μM and 1 μg/ml, respectively. Sclerostin derivedfrom chlorate-treated cells as described in Example 7 (50 μl) was addedto each well, along with 50 μl of either TPST1 and PAPS (“In vitromodified”), or “Unmodified” control of either TPST1 without PAPS or PAPSwithout TPST1. The plate was incubated at 37° C. for 1 hr. Thesclerostin binding assay was performed as described in Example 7.

The results are shown in FIGS. 6 (“Unmodified” is PAPS only) and 7(“Unmodified” is TPST1 only). As shown therein, treatment of theunsulfated sclerostin with TPST1 and PAPS (causing sulfation of thesclerostin) led to greater binding of the alkaline phosphatase-LRP5fusion protein than the unsulfated sclerostin treated with PAPS alone orTPST1 alone. This further confirms that sulfated sclerostin has greaterbinding to LRP5 than unsulfated sclerostin.

Example 9 Analysis of Several Wnt Pathway Proteins for Sulfation SitesUsing Sulfonator

The sulfonator program was used with a variety of different proteinsinvolved in Wnt signaling including members of the Disheveled, Frizzledand Dkk families, as well as the LRP5 and LRP6 receptors. The sequencestested as well as the particular sites where sulfonation sites arepredicted to be located are given below:

Dishevelled (Dvl)

Human Dvl1 O14640 IIYHMDEEE Position 8 (SEQ ID NO: 13) Mouse Dvl1 P51141IIYHMDEEE Position 8 (SEQ ID NO: 14) Human Dvl2 O14641 No site predictedMouse Dvl2 Q60838 No site predicted Human Dvl3 Q92997 No site predictedMouse Dvl3 Q61062 No site predictedConclusion:A potential tyrosine sulfation site was identified by Sulfinator in theDIX region of Dvl 1. The sequence is sufficiently conserved that it isidentical in both human and mouse proteins. However, if Dishevelled isnot processed through the Golgi apparatus, it will not be exposed to aTPST enzyme and will not be sulfated.Dickkopf (Dkk)

Human Dkk1 O94907 DNYQPYPCAEDE Position 83 (SEQ ID NO: 1) Mouse Dkk1O54908 DNYQPYPCAEDE Position 84 (SEQ ID NO: 15) DLDNYQPYPPosition 81 (SEQ ID NO: 16) (overlapping Tyr sites in mouse Dkk1)Human Dkk2 Q9UBU2 No site predicted Mouse Dkk2 Q9QYZ8 No site predictedHuman Dkk3 Q9UBP4 No site predicted Mouse Dkk3 Q9QUN9 No site predictedHuman Dk4 Q9UBT3 No tyrosines Mouse Dkk4 Q8VEJ3 No site predictedConclusion:The Tyr83 site in human Dkk1 is adjacent to but not part of the firstCysteine Rich Domain (CRD) of Dkk-1 and is found in both the human andmouse versions of Dkk1.Kremen (Kr)

Human Kr1 Q96MU8 GNNFDYWKYGEA Position 175 (SEQ ID NO: 17) PDYWKYGEASSPosition 178 (SEQ ID NO: 18) Mouse Kr1 Q99N43 No site predictedHuman Kr2 Q8NCW0 No site predicted Mouse Kr2 Q8K1S7 No site predictedFrizzled (Fz)

Human Fz1 Q9UP38 No site predicted Mouse Fz1 O70421 No site predictedHuman Fz2 Q14332 No site predicted Mouse Fz2 Q9JIP6 No site predictedHuman Fz3 Q9NPG1 No site predicted Mouse Fz3 Q61086 No site predictedHuman Fz4 Q9ULV1 No site predicted Mouse Fz4 Q61088 No site predictedHuman Fz5 Q13467 No site predicted Mouse Fz5 Q9EQD0 No site predictedHuman Fz6 O60353 ITSHDYLGQETLTEIQ Position 580 (SEQ ID NO: 19) Mouse Fz6Q61089 IADHDYLGQETSTEV Position 580 (SEQ ID NO: 20) Human Fz7 O75084No site predicted Mouse Fz7 Q61090 No site predicted Human Fz8 Q9H461No site predicted Mouse Fz8 Q61091 No site predicted Human Fz9 O00144No site predicted Mouse Fz9 Q9R216 No site predicted Human Fz10 Q9ULW2No site predicted Mouse Fz10 Q8BKG4 No site predictedConclusion:No sites were identified by the Sulfonator program for human Fz1, Fz2,Fz3, Fz4, Fz5, Fz7, Fz8, Fz9 and Fz10 proteins. It should be noted thatPosition 580 of the Fz6 is in the cytoplasmic domain.LRP Receptors

Human LRP5 O75197 AIAIDYDPLEG Position 380 (SEQ ID NO: 21)PHSQYLSAEDSCPPSP Position 1583 (SEQ ID NO: 22) Mouse LRP5 Q91VN0AIAIDYDPLEG Position 379 (SEQ ID NO: 23) PHSQYLSAEDSCPPSP Position 1582(SEQ ID NO: 24) Human LRP6 O75581 No site predicted Mouse LRP6 O88572TSDVNYDSEPVPPPTP Position 1562 (SEQ ID NO: 25) Human LRP4 O75096No site predicted Mouse LRP4 Q8V156 No site predictedNeither LRP4 nor LRP6 are predicted to have sulfonation sites located onthe extracellular portion; only the LRP5 receptor seems to have a sitein the extracellular portion. Due to its extracellular location, theparticular LRP5 site should be exposed and available for modification aspart of the second YWTD domain (SEQ ID NO:26) (located at positions341-602).

Example 10 Inhibition of Wnt by Sulfated Dkk1

Native Dkk1 (having some sulfation) was untreated, or treated witheither TPST1, TPST2 or both TPST1 and TPST2 to increase the sulfation ofthe Dkk1. These four Dkk1 preparations were used at three concentrationsin a cell-based luciferase assay using the Wnt reporter cellsessentially as described in U.S. Patent Publication 2006/0198791 todetermine the effect of sulfation on the ability of Dkk1 to inhibit Wntsignaling. Briefly, the cells were seeded in 96-well microtiter platesat 2×10⁵ cells per well, in 50 μl assay medium. After incubation at 37°C. in a 5% CO₂ atmosphere overnight, 50 μl DMEM was added to each well.Wnt3a was then added, to a final concentration of 300 ng/ml, followed byaddition of either buffer, sulfated Dkk1 or unsulfated Dkk1, where Dkk1was added to a final concentration of 0.4, 0.133 or 0.044 μg/ml. Theplates were then incubated at 37° C. in a 5% CO₂ atmosphere for 6 h.Luciferase substrate was then added and chemiluminescence was measured.Each treatment was performed in duplicate.

The results are shown in FIG. 8. As shown therein, the threeTPST-treated Dkk1 preparations inhibited Wnt activity to a greaterdegree than native Dkk1 alone. This establishes that sulfated Dkk1inhibits Wnt signaling to a greater degree than unsulfated Dkk1.

FIG. 8 also shows that the TPST1-treated Dkk1 inhibited Wnt activitymore than the other TPST-treated Dkk1 preparations, indicating thatTPST1 is a more effective enzyme for sulfating Dkk1 than TPST2.

Example 11 Inhibition of Wnt by Unsulfated and Sulfated Dkk1

Dkk1 was prepared in insect cells that were either treated or untreatedwith chlorate, similar to the preparation of sclerostin described inExample 7 above. The treated (unsulfated) or untreated (sulfated) Dkk1was then used in an assay for Wnt activity as described in Example 10.

Results are shown in FIG. 10. Similar to Example 10, sulfated Dkk1exhibited greater inhibition of Wnt activity than unsulfated Dkk1.

Example 12 Sclerostin Peptides with Sulfation Sites ReverseSclerostin-Mediated Wnt Inhibition

The following peptides were synthesized by standard methods:

(SEQ ID NO: 27) P43 = H-P-E-L-G-E-Y-P-E-P-P-P-E-L-E-C-NH₂(SEQ ID NO: 28) P213 = H-C-R-S-A-K-A-N-Q-A-E-L-E-N-A-Y-OH

These peptides have the sequence of human sclerostin at amino acids37-52, where the sulfation site is at amino acid 43 (P43), and aminoacids 198-213, where the sulfation site is at amino acid 43 (P213),using the UniProtKB Accession No. Q9BQB4 of unprocessed sclerostin asreference points.

To determine the effect of P43 and P213 on the ability of sclerostin toinhibit Wnt signaling, a cell based reporter assay was performed using293T cells. Briefly, 293T cells were seeded into 24-well plates 24 hoursprior to transfection. Each well was then transfected with 0.25 μg totalDNA including the constructs of 0.075 μg Lef-Luc, 0.025 μg CMV-Lef1,0.05 μg GFP and 0.1 μg LacZ according to the Lipofectamine™ manual(Invitrogen, Carlsbad Calif.). One day after transfection, growth mediumin each well was replaced with 200 μl DMEM containing 25 mM HEPES. Wnt3awas then added to a final concentration of 300 ng/ml, followed byaddition of either buffer, sclerostin or sclerostin plus peptides, wheresclerostin was added to a final concentration of 10 μg/ml and peptideswere added to a final concentration of 80 μM. Each treatment wasperformed in duplicate. The plates were then incubated at 37° C. in a 5%CO₂ atmosphere for 6 h. Lysis buffer (Roche, Indianapolis Ind.) wasadded, and 40 μl lysate from each well was transferred to a differentwell of the microtiter plate. Background GFP fluorescence was measured,then luciferase substrate (Roche) was added and chemiluminescence wasmeasured with a plate reader.

The results of these assays are shown in FIG. 11. As shown therein, eachpeptide partially reversed sclerostin-mediated Wnt inhibition. It ishypothesized that the peptides compete with sclerostin for LRP binding,thus preventing sclerostin from inhibiting Wnt signaling.

Example 13 Inhibition of Sclerostin Binding by Sulfated and UnsulfatedPeptides

The ability of sulfated or unsulfated P43 and P213 peptides described inExample 12 above were tested for their ability to inhibit sclerostin-LRPbinding. For this study, unsulfated P43 is PRN8829, sulfated P43 isPRN8830, and unsulfated P213 is PRN8831. PRN8830 was sulfatedchemically, and, when PRN8831 was sulfated, it was sulfated using TPST1and/or TPST2 essentially as described in Example 1.

Sclerostin binding to LRP in the presence or absence of the sulfated orunsulfated peptides, at 667 μM peptide concentration, was tested usingthe binding assay essentially as described in Example 3.

The results of these assays are shown in FIG. 12. As shown therein, thesulfated and unsulfated peptides inhibited binding of sclerostin to LRP.

In view of the above, it will be seen that several objectives of theinvention are achieved and other advantages attained.

As various changes could be made in the above methods and compositionswithout departing from the scope of the invention, it is intended thatall matter contained in the above description and shown in theaccompanying drawings shall be interpreted as illustrative and not in alimiting sense.

All references cited in this specification are hereby incorporated byreference. The discussion of the references herein is intended merely tosummarize the assertions made by the authors and no admission is madethat any reference constitutes prior art. Applicants reserve the rightto challenge the accuracy and pertinence of the cited references.

What is claimed is:
 1. A composition comprising a peptide comprisingamino acids and/or amino acid analogs, the peptide comprising acontinuous sequence of a sclerostin fragment comprising Tyr43 or Tyr213,wherein the sclerostin fragment is less than about 75 amino acids, andwherein the peptide is sulfated at the amino acid or amino acid analogcorresponding to Tyr43 or Tyr213 of sclerostin.
 2. The composition ofclaim 1, comprising two or more of said peptides covalently bound toeach other.
 3. The composition of claim 1, wherein the peptide iscapable of inhibiting sclerostin binding to an LRP.
 4. A compositioncomprising a peptide comprising less than about 75 amino acids and/oramino acid analogs including an amino acid or amino acid analog capableof being sulfated, wherein the composition is capable of inhibitingsclerostin binding to an LRP, wherein the tyrosine or tyrosine analog issulfated.
 5. The composition of claim 4, wherein the peptide comprises asequence at least about 50% homologous to a continuous sclerostinfragment comprising Tyr43 or Tyr213, wherein the peptide comprises atyrosine or tyrosine analog at the position analogous to the Tyr43 orTyr213 of human sclerostin.
 6. A composition comprising a peptidecomprising less than about 75 amino acids and/or amino acid analogsincluding an amino acid or amino acid analog capable of beingpost-translationally sulfated, wherein the composition is capable ofinhibiting binding of a protein ligand comprising a sulfation site toits binding partner, and wherein the amino acid or amino acid analog issulfated.
 7. The composition of claim 6, wherein the sulfation site onthe protein ligand is a tyrosine.
 8. A composition comprising a peptideselected from ELGEYPEPPPELENNK (SEQ ID NO: 5), KANQAELENAY (SEQ ID NO:8) or a combination thereof, wherein the peptide is not sulfated.
 9. Acomposition comprising a peptide comprising amino acids and one or moreamino acid analogs, the peptide comprising a continuous sequence of asclerostin fragment comprising Tyr43 or Tyr213, wherein the sclerostinfragment is less than about 75 amino acids.
 10. The composition of claim9, wherein the peptide is sulfated at the amino acid or amino acidanalog corresponding to Tyr43 or Tyr213 of sclerostin.
 11. Thecomposition of claim 9, wherein the peptide is not sulfated at the aminoacid or amino acid analog corresponding to Tyr43 or Tyr213 ofsclerostin.
 12. The composition of claim 9, wherein the peptide isselected from ELGEYPEPPPELENNK (SEQ ID NO: 5), KANQAELENAY (SEQ ID NO:8) or a mixture thereof.
 13. The composition of claim 12, wherein thepeptide is sulfated at the amino acid or amino acid analog correspondingto Tyr43 or Tyr213.
 14. The composition of claim 9, wherein the peptideis not sulfated at the amino acid or amino acid analog corresponding toTyr43 or Tyr213.
 15. A composition consisting of a peptide comprisingamino acids and/or amino acid analogs, the peptide comprising acontinuous sequence of a sclerostin fragment comprising Tyr43 or Tyr213,wherein the sclerostin fragment is less than about 75 amino acids,wherein the peptide is sulfated at the amino acid or amino acid analogcorresponding to Tyr43 or Tyr213 of sclerostin, and wherein the peptideis capable of inhibiting sclerostin binding to an LRP.
 16. Thecomposition of claim 15, wherein the peptide is selected fromELGEYPEPPPELENNK (SEQ ID NO: 5), KANQAELENAY (SEQ ID NO: 8) or a mixturethereof.